Early initiation of antiretroviral therapy (eiART) can improve clinical outcomes for persons with HIV and reduce onward transmission risk. Baseline drug resistance testing (bDRT) can inform regimen selection upon subsequent treatment initiation. We examined the uptake of eiART and bDRT within 3 months and 30 days of HIV diagnosis.We analyzed a population-based sample from the San Francisco Department of Public Health HIV/AIDS Case Registry of newly-diagnosed HIV/non-AIDS individuals between 2001 and 2015 who received care at publicly-funded facilities .Uptake of eiART within 3 months of diagnosis increased significantly from 2001 to 2015 (p<0.001), peaking at 74% in 2015. bDRT uptake also increased significantly (p<0.001), peaking at 55% in 2012. eiART uptake was observed to be significantly associated with gender, age, race/ethnicity and transmission risk. There were no significant differences observed in demographic and risk characteristics of persons receiving bDRT in the more recent years. Of 990 persons diagnosed between 2010 and 2015, eiART uptake within 30 days of diagnosis increased from 13% to 38% (p<0.001); bDRT uptake increased from 35% to 39% but the change was not significant (p = 0.141).Observed increases in eiART and bDRT uptake from 2010 to 2015 may reflect the adoption of treatment as prevention and a local public health policy statement in 2010 recommending treatment initiation at time of diagnosis irrespective of CD4 count. Concerns about stigma may underlie disparities in eiART, however such concerns would not bear as directly on a provider-initiated laboratory test like bDRT. Treatment guidelines revisions were revised to recommend ART initiation at CD4 <350 in 2007 and CD4 <500 in 2009.[HIV treatment guidelines in the US have evolved greatly over the past decade. Historically, antiretroviral therapy (ART) initiation was based on CD4 T cell counts. Prior to 2007, ART initiation was recommended when CD4+ lymphocyte count (CD4 count) dropped below 200 cells/mm in 2009.Informed by clinical trials demonstrating starting ART early improves clinical outcomes for persons with HIV and reduces onward transmission risk, national treatment guidelines have recommended early initiation of ART (eiART) irrespective of CD4 count since 2012. Prior toAdditionally, HIV-1 drug resistance testing (DRT) has long been recommended for the clinical management of patients failing treatment. When a regimen change is indicated due to virologic failure, DRT can guide the selection of drugs effective against the HIV strains a patient carries. Improvements in short-term virologic response to treatment when DRT results are available to clinician have been documented.DRT during early stages of infection has additional benefits. Baseline DRT (bDRT) upon diagnosis and prior to ART initiation can detect transmitted drug resistance mutants present early in the course of infection that decrease over time but might still persist at undetectable levels in the absence of ART.,7 bDRT wThe present analysis examined the uptake of eiART and bDRT within both 3 months and 30 days of HIV diagnosis. We also assessed demographic, risk and clinical characteristics of newly-diagnosed persons with eiART and bDRT uptake.3 and the absence of an AIDS-indicator condition at time of diagnosis. Receipt of care was defined as having either a CD4 count or an HIV viral load laboratory result. Data were obtained from the San Francisco Department of Public Health HIV/AIDS Case Registry, and the AIDS Research Institute (ARI) University of California, San Francisco (UCSF) Laboratory of Clinical Virology (LCV) which performs drug resistance testing for all local publicly-funded clinics.We analyzed a population-based sample of San Francisco residents newly-diagnosed with HIV/non-AIDS from 2001 through 2015 who received care at publicly-funded facilities. HIV/non-AIDS was defined as CD4 count ≥200 cells/mmPersons not ART-naïve at time of diagnosis were excluded from all analyses. ART initiation date was determined based on the prescription date indicated in the medical records. DRT date was based on the specimen collection date. Two definitions of eiART were used: 1) initiating ART within 3 months of diagnosis; and 2) initiating ART within 30 days of diagnosis. Two definitions of bDRT were used: 1) DRT within 3 months of diagnosis and prior to ART initiation; and 2) DRT within 30 days of diagnosis and prior to ART initiation. The timeframe (3 months or 30 days) was specified in each analysis. Since recommendations for eiART upon diagnosis began in San Francisco in 2010 and nationally in 2012, we assessed eiART and bDRT within 30 days of diagnosis for persons diagnosed between 2010 and 2015.Demographic and risk characteristics included gender, age, race/ethnicity and HIV transmission risk category. Clinical characteristics included HIV diagnosis date, first DRT date and ART initiation date. Cases were characterized by diagnosis year and stratified by treatment guideline eras. Diagnosis years were grouped into eras based on treatment guidelines or when a significant local policy on ART was implemented. Era 1 spanned from 2001 through 2003, Era 2 from 2004 through 2006, Era 3 from 2007 through 2009, Era 4 from 2010 through 2012, and Era 5 from 2013 through 2015.eiART and bDRT temporal trends and associations with demographic, risk and clinical characteristics were assessed by Chi-Square and Fisher’s Exact tests, and multivariable logistic regression. The study received approval from the UCSF Institutional Review Board. A waiver of informed consent was granted with the stipulation that all identifiable patient data, e.g., clinical results, and demographic and risk characteristics, would be maintained securely behind the mandatory data firewall of the San Francisco Department of Public Health.A total of 3,124 persons newly-diagnosed with HIV/non-AIDS in San Francisco between 2001 and 2015 received care at publicly-funded facilities. These included 723 persons diagnosed in Era 1, 753 persons in Era 2, 658 persons in Era 3, 565 persons in Era 4 and 425 persons in Era 5. Demographic and risk characteristics, stratified by eras, are shown in Overall, there were 729 persons (23%) receiving eiART and 836 persons (27%) receiving bDRT within 3 months of diagnosis. The proportion of individuals by year is presented in The proportion of cases with eiART and bDRT within 3 months of HIV diagnosis stratified by eras is shown in Demographic and risk characteristics of persons receiving eiART and bDRT within 3 months of diagnosis, stratified by eras, are presented in Of the 990 persons diagnosed between 2010 and 2015, 237 persons (24%) received eiART and 361 persons (36%) received bDRT within 30 days of diagnosis. The proportion of individuals receiving eiART increased significantly from 13% in 2010–2012 to 38% in 2013–2015 (p<0.001). The proportion of individuals receiving bDRT rose from 35% in 2010–2012 to 39% in 2013–2015 but the increase was not significant (p = 0.141). Demographic and risk characteristics of persons receiving bDRT are detailed in The proportion of persons newly-diagnosed with HIV/non-AIDS who initiated ART within 3 months of diagnosis increased significantly from 2004 through 2015. The substantial increase in eiART uptake observed in the more recent years likely reflect adoption of revised local and national guidelines, as well as the strategy of treatment as prevention.,9 The deART has been shown to increase survival and decrease morbidity, and the 2010 San Francisco universal treatment policy was influenced by findings released the previous year demonstrating lower mortality risk among patients initiating ART at higher CD4 counts.,9 HowevebDRT within 3 months of diagnosis increased steadily from 2004 through 2012, with a slight decline in 2013 to 2015, always remaining less than 40%. bDRT was less common in the early part of the decade, likely because the national guidelines at the time did not include bDRT. The finding that fewer than half of cases had bDRT was unexpected, especially in the more recent years when baseline testing recommendations had been placed for several years and testing had become available to San Francisco residents regardless of insurance coverage. Potential barriers to resistance testing include lack of familiarity with how resistance testing is ordered, especially in testing centers where HIV is diagnosed but treatment is not routinely provided. Financial barriers to resistance testing would still occur if the person was not a resident of San Francisco or if they migrated out of San Francisco before resistance testing was ordered. Ethnic disparities in treatment access and resistance testing were present in 2001–2003 and lessened in the following eras as treatment and resistance testing scaled up. While eiART involves multiple opportunities for stigma to create disparities, bDRT is a laboratory test that poses little additional risk of stigma or unintended disclosure.The proportion of individuals receiving both bDRT and eiART within 3 months of diagnosis was highest in 2013–2015, which likely reflects the combined impact of revisions to the treatment and drug resistance testing guidelines. Both eiART and bDRT within 30 days of diagnosis increased between 2010 and 2015. The increase in bDRT was driven primarily by the corresponding increase in eiART. Local and national guideline revisions in recent years recommending treatment initiation upon diagnosis likely propelled the observed increases.Even for cases receiving bDRT who did not initiate treatment early, baseline genotyping can inform regimen selection upon subsequent ART initiation because the abundance of resistant viruses may decline over time in the absence of selective drug pressure but may be still present in viral archives. Upon ART initiation, even low levels of resistant viruses may increase risk of treatment failure.–13 bDRT Associations observed among the study sample may differ from individuals receiving care at private facilities. Nonetheless, since publicly-funded facilities provide care for three-quarters of newly-diagnosed cases annually, these results are representative of a majority of the recently-diagnosed HIV population in San Francisco. Data forBoth eiART and bDRT were adopted by local clinical practices in San Francisco as early as 2004, before the U.S. treatment guidelines included these recommendations on a national basis. Further analyses are needed to evaluate the benefits of the combined practice of eiART and bDRT in ultimately improving clinical outcomes.


To describe the clinical decisions taken for patients failing on treatment and possible implementation leakages within the monitoring cascade at a large urban HIV Centre in Kampala, Uganda.  As per internal clinic guidelines, VL results >1,000 copies/ml are flagged by a quality assurance officer and sent to the requesting clinician. The clinician fills a “decision form” choosing: (1) refer for adherence counselling, (2) repeat VL after 3 months, and (3) switch to second line. We performed data extraction on a random sample of 100 patients with VL test >1,000 copies/ml between January and August 2015. For each patient, we described the action taken by the clinicians. st-line ART, 19% on 2nd-line, median CD4 count was 249 cells/µL (IQR: 145–390), median log10 VL 4.42 (IQR: 3.98–4.92). Doctors' decisions were; refer for adherence counseling 49%, repeat VL for 25%, and switch to second line for 24% patients. Forty-one percent were not managed according to the guidelines. Of these, 29 (70.7%) were still active in care, 7 were tracked  and 5 patients were not tracked.  Of 6,438 patients with VL performed, 1,021 (16%) had >1,000 copies/ml. Of the 100 (10.1%) clinical files sampled, 61% were female, median age was 39 years (IQR: 32–47), 81% were on 1 Despite the implementation of internal systems to manage patients failing ART, we found substantial leakages in the monitoring “cascade”. Additional measures and stronger clinical supervision are needed to make every test count, and to ensure appropriate management of patients failing on ART. Remarkable progress has been made in the scale-up of antiretroviral therapy (ART) in low and middle-income countries (LMICs). Currently, approximately 21.7 million people are receiving ART globally, including 15 million people in sub Saharan Africa [In order for HIV treatment to be effective in restoring immune-functionality, ART should control viral replication and patients should achieve viral suppression. Therefore, periodic viral load (VL) testing is considered the gold standard approach for ART monitoring in HIV positive patients and in countries where VL testing is available. The most common cause for not achieving viral suppression is poor adherence, however, suboptimal drug concentrations due to malabsorption or drug–drug interaction, and transmitted drug resistance can ultimately contribute to viral replication .In the 2013 consolidated ART guidelines , WHO recDelayed switching to second line treatment in patients with treatment failure increases risk of morbidity and mortality , 8. AddiWe investigated clinical decision-making and subsequent management of a sample of patients with VL >1,000 copies/ml after the implementation of routine VL monitoring at a large urban HIV Centre in Kampala, Uganda.The Infectious Diseases Institute (IDI), Makerere University is a center of excellence  for HIV In prevailing clinic guidelines in 2015, VL results >1,000 copies/ml were flagged by a quality assurance officer and managed through a multidisciplinary  weekly “switch-meeting”, previously described . At thesThis was a retrospective study of 100 randomly sampled HIV infected patients on ART who had a VL results >1,000 copies/ml from January to August 2015 at IDI.Data from patient's clinical charts and the clinic electronic database  were valActions taken by the clinicians for each patient were summarized.Statistical analysis was performed using Stata version 12.2 .The study and protocol for retrospective use of routinely collected data at IDI was reviewed and approved by the ethics and protocol review committees of Makerere University, Faculty of Medicine Research and Ethics Committee  and the Uganda National Council for Science and Technology . Patient's information was analyzed after anonymization by removal of unique personal identifiers and as such, they did not provide written or verbal consent as per the protocol procedures.µL (IQR: 145–390), the median log10 VL 4.42 (IQR: 3.98–4.92) and a majority (81%) were on 1st-line ART. Between January and August 2015, 6,438 patients had their VL done, 1,021 (16%) had detectable viral loads (VL) . Of the sampled 100 (10.1%) patients, majority (61%) were female, the median age was 39 years (IQR: 32–47), the median CD4 count was 249 cells/nd line ART after a median time of 189 days (IQR: 132–231).Of the 25 patients who were recommended for a repeat viral load, 18 (72%) had the test repeated, of which 17 (94%) had a VL >1,000 copies/ml and of these 10 (59%) were switched to 2nd line after a median time of 214 days (IQR 177–238).Forty-two (86%) of the 49 patients who were referred for adherence counseling, were counseled and after 3 months, 31 of them (74%) were recommended for a repeat VL; 30 (97%) had their VL retested among which 21 (70%) had VL >1,000 copies/ml; 15 (71%) were also switched to 2As shown in the greyed boxes in In this study we evaluated the management of patients with suspected treatment failure as indicated by their detectable viral loads in our program. In 59% of the cases, patient management adhered to the prevailing clinic guidelines despite introduction of a quality assurance officer to track and coordinate detectable VL results. However, prior to the implementation of routine VL monitoring, we conducted a similar analysis on patients who receive clinician driven VL testing due to a suspicion of treatment failure and we found high (96%) adherence to the guidelines .Our study likely indicates that introduction of routine VL testing increased volumes of patients with detectable VL to manage which placed a strain on the clinic system for managing patients with treatment failure. Notably, we observed considerable delays in switching patients to second line, but also for patients that were correctly managed; this was mainly due to delays in discussing the cases at the weekly switch meeting, due to the sudden increase in numbers of patients detected, with viral failure.Our study contributes to the literature by delineating the exact steps within the cascade where nonadherence to guidelines occurs and that can be used to develop interventions. Although no formal assessment was conducted to ascertain the relative contribution on possible factors responsible for the leakages in our monitoring cascade, we believe that introduction of a quality control system to ensure timeliness in implementing patient management decisions along the cascade would be beneficial.Substantial leakages and delays in the cascade for managing patients with detectable VL still exist. In order to make every patients' VL test count, additional measures including substantial investment in strengthening internal health quality control systems and structures as well as capacity building among medical staff to strengthen clinical care decisions and closer monitoring of patients for timely decision making along the care cascade should be implemented.


Female sex workers (FSWs) play a key role in HIV transmission in West Africa, while they have limited access to antiretroviral therapy (ART). In line with UNAIDS recommendations extending ART to all HIV-infected individuals, we conducted this demonstration project on immediate treatment as prevention (TasP) among FSWs in Cotonou, Benin. We report data on treatment response and its relation to adherence, as well as on ART-resistant genotypes.Complete follow-up varied between 12 and 24 months. At each three-monthly visit, a questionnaire was administered, clinical examinations were carried out and blood samples collected. Adherence to treatment was estimated by self-report. Viral RNA was genotyped at baseline and final visits for drug resistance. Generalized estimating equations for repeated measures with a log-binomial link were used to analyze time trends and the association between adherence and virological response to treatment.One-hundred-seven HIV-positive and ART-naive FSWs were enrolled; 59.8% remained in the cohort till study completion and 62.6% had a final visit. Viral load<1000 (below quantification limit [<50]) was attained in 73.1% (64.6%) of participants at month-6, 84.8% (71.2%) at month-12, and 80.9% (65.1%) at the final visit. The proportion of women with suppressed (below quantification limit) viral load increased with increasing self-reported adherence (p = 0.06 (0.003), tests for trend). The proportion of participants with CD4≤500 also decreased drastically throughout follow-up (p < .0001). Twelve participants exhibited ART-resistant genotypes at baseline, but only two at their final visit.Our findings indicate that TasP is widely accepted among FSWs in Cotonou and could be implemented with relative success. However, due to mobility in this population, follow-up was sub-optimal, suggesting that large geographical coverage of FSW-friendly clinics is needed for sustained treatment implementation. We also fell short of the UNAIDS objective of 90% viral suppression among treated patients, underlining the need for better adherence support programs. Western and central Africa is the second most affected region by the HIV epidemic with an estimated 5 million people living with HIV (PLHIV) at the end of 2018 . Yet, HIV testing and antiretroviral therapy (ART) coverage in this region are among the lowest worldwide , play a Recruitment was conducted in pre-specified areas of Greater Cotonou (Cotonou and close suburbs) in order to approximately meet target sample sizes of 250 for the PrEP arm, and 100 for the TasP arm. After a run-in phase for community preparedness and the development of a specific education program on adherence, FSWs from the catchment areas were invited to attend the “Dispensaire IST”, a clinic dedicated to FSWs for HIV and sexually transmitted infections (STIs) care in Cotonou, for HIV testing. HIV-positive FSWs not known to be on ART were then invited to participate in the TasP component of the study and received a first-line ART regimen as per the Benin guidelines. Recruitment took from October 2014 to December 2015, followed by an additional one-year follow-up till December 2016, for a total follow-up varying between 12 and 24 months, depending on when a given participant was recruited in the study. A short-term follow-up visit to assess adherence and drug adverse events was scheduled at day 14 with subsequent visits being scheduled quarterly. At baseline and during follow-up visits, clinical examinations were carried out, vaginal and blood samples collected for STIs testing, CD4 count, and viral load quantification. Adherence was assessed during follow-up visits by self-report through a questionnaire. Participants who came back to the clinic for withdrawal were invited to undergo all the procedures of a final visit, while those who stopped participating without informing the clinic, were located by field workers and reasons for dropping out documented.The study population consisted of professional FSWs (e.g. whose main revenue comes from sex work) 18 years or older, practicing in the pre-defined catchment areas. Those eligible for TasP had to be HIV-1 positive at screening, re-confirmed on a second test at the recruitment visit, and being treatment naïve. The only exclusion criterion was HIV-2 or dual HIV-1/2 infection.Several socio-demographic and sex work characteristics including nationality, age, marital status, education, parity, activities beside sex work, duration in sex work and monthly income were assessed through a questionnaire at baseline. Sexual behaviour including number of clients in the last two weeks, having a regular sexual partner and condom use with both clients and regular partners were assessed through the questionnaire at baseline and during follow-up. In order to assess adherence, at each visit, questions were asked on the number of pills missed in the last month.Trichomonas vaginalis; and abnormal vaginal flora using the Nugent score for the diagnosis of bacterial vaginosis. Participants also underwent testing for Neisseria gonorrhoeae and Chlamydia trachomatis on cervical swabs at recruitment and every six months using the BD Probetec NG/CT assay . Additional tests including glycaemia, blood creatinine serum levels , and alanine aminotransferase serum levels (liver function), as well as Chest-X ray prior to ART initiation were conducted, and complete blood count carried out using standard assays. Blood samples were collected and stored at screening and final visits for future drug resistance genotyping. In the context of the present study, CD4 count was monitored at screening and every three months using a CyFlow Counter , whereas viral load was quantified at the screening visit and then every six months using the NucliSens EasyQ equipment . The technique allows the quantification of a minimum of 50 copies/ml of HIV-1 RNA in 0.5 ml plasma Reviewers' comments:Reviewer's Responses to QuestionsComments 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: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: Yes**********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: No**********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 AuthorPlease 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: General comments:The authors report on the results of a Treatment as Prevention demonstration study among a cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin.Their findings on acceptability, retention and viral load provide important and relevant information which should be taken into account when implementing TasP among key populations in Africa.The general methods seem adequate. Statistical methods and analysis however should be verified. Some of the statistical tests used are not mentioned. The authors use generalized estimating equations to analyse time trends and the association between adherence and virological response however the results of the model are not clear and complete. It is also a pity that no attempt for identifying or adjusting for confounders have been made.Specific comments:Introduction- P5, line 59: “an estimated 6.1 million people living with HIV at the end of 2016”Please update the numbers and add a reference.- P5, line 73: “.(14)”Please correct this typo.Methods- P.6, line 90: “FSW not known to be HIV-positive on ART”This expression is not clear, why the specification “on ART”?. Do you mean “FSW not known to be HIV-positive” ?- P. 8, lines 130-132. Syphilis tests.There may be some confusion when using the terms “rapid test” and “RPR”. Can you make it clear that the rapid is a treponemal, and the RPR is a nontreponemal test. One could also argue that one positive RPR test is not sufficient to detect active syphilis. According international guidelines there is a need of a fourfold change in titer, equivalent to a change of two dilutions to demonstrate an active infection.Results- P. 13, line 237: “13.2 ± 7.7”Please specify that 7.7 is a standard deviation (I guess)- P. 13, line 241: “the main reasons for not completing final visits”The reason for not completing final visits is not the main issue I guess, but the reason for not being retained on ART, whether they come for a final visit or not.- P. 15, line 277: “were infected or had a history of NG/CT”Was this a history of an infection “ever”? It would make more sense to separate the biological results (STI at baseline) and the interview data “did you ever had an STI”, because they are two separate things.- P .20, table 3 “Mean”Please add “Mean CD4” for clarity in the title and the column head- P. 20, table 3: p-valuesThe statistical methods (tests) for this table are not shown, please indicate which tests were used to obtain both type of p-values , and explain in the methods.- P. 23, table 4: p-valuesIdem as previous comment.- P. 24, lines 363-367: “the proportion … increased with increasing adherence (p=0.06 and 0.003)”The results in the table do not confirm this statement: the proportion of visits where viral load was suppressed was 83.2% for the group with 90% adherence and 84.2% for the group 75-89% adherent.- P. 24, table 5: results GEEThe table is quite difficult to read. The column with the head “Ratio” seems to present prevalences, not prevalence ratios. A prevalence is also a ratio in theory, but in this situation the term “ratio” adds to confusion.- P. 24, table 5, p for trend (p=0.06 and 0.003)Which test has led to these p’s? It is curious to see a p for trend of 0.06, when the PR are exactly the same in all adherence categories (with the exception of the reference category). Idem for the p for trend 0.03 for the outcome “undetectable”, where PR go from 3.2 for the group with the best adherence, vs 3.3 for the group with adherence 75% and 50%.- P. 24, table 5: results GEEThe table only show the PR of the virological response per adherence level, the time factor is not presented. Have the authors considered to check potential confounders including age, behaviour factors such as condom use ? This may provide interesting alternative explanations for the relation adherence/virological outcome.- P. 28-30, table 6Please revise lay-out of the table, as the text in the lines is not positioned in a standardized way, which make the table difficult to read.Discussion- P. 31, line 440-441Revise structure. The logical flow can be improved by replacing: “since half of them are not Beninese, and even when they were …”- P. 32, line 453: “mobility”Mobility is indeed be a serious barrier. The authors may discuss here the access of the FSW to ART services if they move to another place, another country.- P. 32, line 466Correct typo: “fact”- P. 34, lines 509-512: “The results revealed … interventions”This paragraph is repeated later in the discussion. It is a general conclusion and should be moved to the end of the paper.- P. 35, lines 527-529The authors should be cautious in their conclusions about the association between self-reported adherence and viral load, taking into account the results (see higher)Reviewer #2: General comments1. Although the study describes a TasP/PrEP demonstration project, limited data are provided about PrEP2. The accession numbers of the nucleotide sequences generated in this project are not provided3. The number of Tables should be reducedPage 26, HIV strains and drug resistance (details in Table 6)1 Authors provide subtype classification twice (i.e. CRF02_AG/CRF02_AG). This should be explained2 The prevalence of resistance mutations should be reported**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  27 Sep 2019Dr Orna Mor, Ph.D. Academic EditorPLoS ONERE: Response to reviewers - Manuscript ID: PONE-D-19-15725Dear Editor,We would like to sincerely thank the editor and the reviewers for the comprehensive evaluation of our manuscript entitled “HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin” that has been submitted for publication in PLOS ONE. We are excited the reviewers found our manuscript to be of significance to the field. Their pertinent comments and suggestions have definitely contributed to strengthen our paper, and as such, we have done our best to address all the editor’s and reviewers’ concerns. These changes are highlighted in the manuscript with the Word “Track changes” function. Since major revisions were brought and as requested in the letter from the academic editor, a “clean” version of the manuscript is also provided. As the revised manuscript also includes two figures, we have uploaded the figure files separately and have written the figure title and legends following the text that explains about the figure. Please find below the comments and questions  followed by our answers and details about the corrections that we performed on the original manuscript. Please note that when we refer to line numbers, those refer to line numbers as read in the clean version of our revised paper without tracked changes.The revised manuscript is now ready for further consideration by PLoS ONE. We sincerely hope that it will now be suitable for publication in your very interesting journal. Best Regards,Mamadou Diallo and Michel Alary Note from the Editor:1) We note that you have indicated that data from this study are available upon request.Response: Indeed, as we mentioned while submitting the manuscript, data are available and can be made accessible. We have now provided a minimal anonymized data set necessary to replicate our study findings. This file uploaded as a supporting Information file can be accessed in the PloS ONE website.Additional Editor Comments (if provided):The authors report on the results of a TasP demonstration study among a small cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin. In general, the study is clear and well written and interesting. However, the number of tables versus size of study is uncomparebly high.2) Table 1 could be removed and explained in the textResponse: We have removed the table 1 and explained the corresponding data in the text (page 12-13: line 230-233). 3) A graph of cd4 and vl versus visits could replace tables 3 and 4. Response: We have replaced tables 3 and 4 by Figures 1 and 2 (2 figures needed to provide enough details) that we have uploaded as separate files. Figure titles and legends are provided in the manuscript itself . We have also slightly modified the text describing these 2 figures to provide some of the actual numbers that were in the table and that are somewhat less clear in a figure format .4) Table 5 is unclearly presentedResponse: We have entirely reformatted table 5, which is now table 2 (see page 21). 5) On the other hand, a table summarizing the prevalence and type of baseline DRM in the different class of drugs versus subtypes is missing. Response: The information on baseline DRM was already present in former Table 6; indeed, we agree it was badly formatted. We have also entirely reformatted this table that is now Table 3 (see page 24-25). In addition, we now provide data on baseline prevalence of DRM in the text .6) The outline of Table 6 is also unclear and if a new summary table is provided- then table 6 can be in appendix. Response: See our response to the previous comment. 7) Please also check if all references are correctly written and according to PLOS policy.Response: We have checked the references and adjusted them to PLOS policy where necessary.Reviewer #1: General comments: 8) The authors report on the results of a Treatment as Prevention demonstration study among a cohort of 107 HIV-infected, treatment naïve, female sex workers in Cotonou, Benin. Their findings on acceptability, retention and viral load provide important and relevant information which should be taken into account when implementing TasP among key populations in Africa. The general methods seem adequate. Statistical methods and analysis however should be verified. Some of the statistical tests used are not mentioned. The authors use generalized estimating equations to analyse time trends and the association between adherence and virological response however the results of the model are not clear and complete. It is also a pity that no attempt for identifying or adjusting for confounders have been made. Response: We thank the reviewer for addressing this aspect of the methodology of data analysis, and his willing to have more information in the methodology. We hereby provide detail information on the methodology and reasons we used for the analysis. Indeed, since we had repeated data, the use of generalized estimating equations with the log –link and the type3 test of Wald seemed to be more appropriate for the analysis of time trend changes as well as the association between viral load and self-reported adherence. While analyzing the association between viral load and self-reported adherence, the final model controlled for time duration since ART initiation. We also initially included potential confounders such as age, country of origin, duration in sex work, having a regular partner (or not), and number of clients (but not reported condom use as it was reported to be extremely high: see Table) in the multivariate model, however, all these variables did not confound the association between self-reported adherence and viral load. Therefore, we did not keep them in the final model in order to maximize statistical power. We have made some changes in the section on statistical methodology to better describe our approach  as well as in the results section when reporting the associations between self-reported adherence and viral load .Specific comments:Introduction9) - P5, line 59: “an estimated 6.1 million people living with HIV at the end of 2016”Please update the numbers and add a reference.Response: We have updated both the numbers and the reference. (Page 5: line 58-60). 10) - P5, line 73: “.(14)”Please correct this typo.Response: Corrected (page 5: line 73).11) Methods- P.4, line 93: “FSW not known to be HIV-positive on ART”This expression is not clear, why the specification “on ART”? Do you mean “FSW not known to be HIV-positive”?Response: We thank the reviewer for pointing out this ambiguity. Indeed, this expression was misplaced. It’s only after the confirmation of the screening test at the Dispensaire IST, that we restrained recruitment to HIV-positive FSWs who were not known to be on ART. Being ART naïve were confirmed following responses to questions, and confirmation the clinic records as the vast majority of HIV-positive FSWs in Cotonou who are on ART receive their treatment at Dispensaire IST). In the manuscript, the deletion at the inappropriate location of the manuscript is on page 6: line 90-91 and the addition is on page 6, lines 93-95. IST (in order to confirm that participants were ART-naïve)12) - P. 8, lines 130-132. Syphilis tests.There may be some confusion when using the terms “rapid test” and “RPR”. Can you make it clear that the rapid is a treponemal, and the RPR is a nontreponemal test. One could also argue that one positive RPR test is not sufficient to detect active syphilis. According international guidelines there is a need of a fourfold change in titer, equivalent to a change of two dilutions to demonstrate an active infection.Response: We have now specified that the rapid test was treponemal and that the RPR was non-treponemal (page 8: Line 130-133). Note also than in cross-sectional studies , it is usual to apply the definition we used for active syphilis. We don not report follow-up data on this issue, but please note that, in addition to not having a single case of active syphilis at baseline, there was no new case of syphilis during follow-up. Results13) - P. 13, line 237: “13.2 ± 7.7”Please specify that 7.7 is a standard deviation (I guess)Response: This is now specified (page 12: line 230). 14) - P. 13, line 241: “the main reasons for not completing final visits”The reason for not completing final visits is not the main issue I guess, but the reason for not being retained on ART, whether they come for a final visit or not.Response: In fact, we cannot know if the women who did not complete follow-up in the study were retained on ART or not. It depends if they accessed ARV clinics after leaving the study (most of them were not in Cotonou anymore and many were in other countries). Therefore, the reasons given in the manuscript are for withdrawing from the study. The wording of this section has also been changed because of the deletion of original table 1 and the addition of new text following a request from the editor (see page 9: 230-236).15) - P. 15, line 277: “were infected or had a history of NG/CT”Was this a history of an infection “ever”? It would make more sense to separate the biological results (STI at baseline) and the interview data “did you ever had an STI”, because they are two separate things.Response: We thank the reviewer for pointing out this mistake. The prevalence we provided was in fact for the actual biological results of the nucleic acid amplification tests for NG and CT and did not include past history collected by questionnaire. This is now corrected . 16) - P .20, table 3 “Mean”Please add “Mean CD4” for clarity in the title and the column headResponse: As per the editor’s request, we have replaced table 3 by figure 1, but mean CD4 count is now specified in this figure’s title and legend (page 19: Line 282-292).17) - P. 20, table 3: p-valuesThe statistical methods (tests) for this table are not shown, please indicate which tests were used to obtain both type of p-values , and explain in the methods. Response: We described the analysis methodology in the method section. For all the analysis, we used generalized estimating equations with the log–link and the type3 test of Wald (see page 11: Lines 197-202), . Note that table 3 has now been replaced by figure 1.18) - P. 23, table 4: p-valuesIdem as previous comment.Response: Same response as to the previous comment. Note also that table 4 has now been replaced by figure 2. (see page 11: Lines 197-202).19) - P. 24, lines 363-367: “the proportion … increased with increasing adherence (p=0.06 and 0.003)”. The results in the table do not confirm this statement: the proportion of visits where viral load was suppressed was 83.2% for the group with 90% adherence and 84.2% for the group 75-89% adherent.Response: We used 4 adherence categories in this analysis and evaluated a test for trend over the 4 categories and these are the p-values we obtained (see our response to comment 8 above). This can happen even if the various categories yield similar prevalence ratios when compared to the reference category as long as the association is strong enough. However, we agree with the reviewer in the fact that it would be important to qualify this statement by evoking the stability of the prevalence ratios as long as the adherence level is >50%. We have thus added a sentence in this regard in the result section . 20) - P. 24, table 5: results GEEThe table is quite difficult to read. The column with the head “Ratio” seems to present prevalences, not prevalence ratios. A prevalence is also a ratio in theory, but in this situation the term “ratio” adds to confusion.Response: As per the editor’s request, we have completely reformatted this table, that is now table 2, and we have changed the column header as suggested by the reviewer. (see page 21-22: line 334-340).21) - P. 24, table 5, p for trend (p=0.06 and 0.003) Which test has led to these p’s? It is curious to see a p for trend of 0.06, when the PR are exactly the same in all adherence categories (with the exception of the reference category). Idem for the p for trend 0.03 for the outcome “undetectable”, where PR go from 3.2 for the group with the best adherence, vs 3.3 for the group with adherence 75% and 50% (page 15: line 334).Response: See our response to comments 8, 17 and 19 above. 22) - P. 24, table 5: results GEEThe table only show the PR of the virological response per adherence level, the time factor is not presented. Have the authors considered to check potential confounders including age, behaviour factors such as condom use? This may provide interesting alternative explanations for the relation adherence/virological outcome.Response: As time since treatment initiation was treated as a confounding factor for the association between adherence levels and viral load and that we were mainly interested by this latter association, we do not present prevalence ratios for this time variable to keep the table as simple as possible for the readers. Concerning the other potential confounders, see our response to comment 8 above.23) - P. 28-30, table 6Please revise lay-out of the table, as the text in the lines is not positioned in a standardized way, which make the table difficult to read.Response: This table has now been entirely reformatted and its number as changed to table 3 (see page 24-26).Discussion24) - P. 31, line 440-441Revise structure. The logical flow can be improved by replacing: “since half of them are not Beninese, and even when they were …”Response: We have reviewed and rephrased the sentence as suggested (page28: line 418-423).25) - P. 32, line 453: “mobility”Mobility is indeed be a serious barrier. The authors may discuss here the access of the FSW to ART services if they move to another place, another country.Response: We added some discussion on this issue . 26) - P. 32, line 466Correct typo: “fact”Response: Corrected (page 29: line 433)27) - P. 34, lines 509-512: “The results revealed … interventions”This paragraph is repeated later in the discussion. It is a general conclusion and should be moved to the end of the paper.Response: We removed this paragraph as suggested by the reviewer . At the same time, since this sub-section now only present limitations, we changed the name of the sub-section “Study strengths and caveats” to “Study limitations”.28) - P. 35, lines 527-529The authors should be cautious in their conclusions about the association between self-reported adherence and viral load, taking into account the results (see higher)Response: We have made some changes in order to be more cautious with this conclusion .Reviewer #2: General comments29) Although the study describes a TasP/PrEP demonstration project, limited data are provided about PrEPResponse: We thank the reviewer for his interest in the PrEP data. Although our project focused on TasP and PrEP, we only report data on TasP in this paper. Indeed, we have already published a paper on the overall resust of our TasP/PrEP study in the Journal of the International AIDS Society (JIAS) (see 20 of the present study or DOI: 10.1002/jia2.25208). The title of this paper is “Early antiretroviral therapy and daily pre-exposure prophylaxis for HIV prevention among female sex workers in Cotonou, Benin: a prospective observational demonstration study. However, the results presented in the present paper include some additional TasP subjects and present analyses that are not included in our JIAS paper.30) The accession numbers of the nucleotide sequences generated in this project are not providedResponse: We perfectly agree with the reviewer that the nucleotide sequences generated in this project should be provided. Unfortunately, we did not think about recording these sequences in Genbank. However, if requested, we can provide s the sequences for verification. 31) The number of Tables should be reducedResponse: This has been done: we went from 6 tables to 3 tables and 2 figures following the editor’s comments. 32) Page 26, HIV strains and drug resistance (details in Table 6)Authors provide subtype classification twice (i.e. CRF02_AG/CRF02_AG). This should be explainedResponse: As to our understanding of the question, the reviewer asks us to explain why we reported the same subtype twice (i.e. CRF02_AG/CRF02_AG). This is a standard terminology for presenting the results of genotyping for drug resistance. Indeed, sequencing the HIV-1 pol and env genes is routinely used to identify mutations associated with resistance to reverse transcriptase (RT) or protease (PR) inhibitors in HIV-1 circulating recombinant forms (CRFs) including CFR02_AG and CRF01_AE. Samples were genotyped at both env and pol regions. When the results yielded identical sequences in the two regions, we reported the same sequence twice (ex.: CRF02_AG/CRF02_AG), otherwise we reported differently (i.e. CRF11-cpx/CRF02_AG). This appears in the revised table 3 (i.e. G/CRF02_AG and CRF11-cpx/CRF02_AG) and shows the different strains in our study.The prevalence of resistance mutations should be reported Response: We now report the prevalence of resistance mutations in the text of the manuscript .AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 25 Oct 2019PONE-D-19-15725R1HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, BeninPLOS ONEDear Dr. Alary,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.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Dec 09 2019 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Orna MorAcademic EditorPLOS ONEAdditional Editor Comments (if provided):Thanks you for submitting the revised manuscript. The previous corrections requested by the reviewers were met. However, there are still few minor remarks on the revised version. I do not find that the correction requested by reviewer 1 is necessary as indeed as stated and discussed t is possible that some of the FSW were on ART previous the study. Therefore I suggest to leave the sentence "HIV positive FSW not known to be on ART"Regarding the remark of reviewer 2- I agree, if it is possible.The followings are few remarks that if corrected, could better clear this paper:Line 139: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT). In Table 1 : N. gonorrhoeae or C. trachomatis. Please choose either NG/CT or N. gonorrhoeae - C. trachomatis throughout the text.Page 9 148-151: nuclisens can officially assess HIV-1 only (not HIV-2). Only one publication (ref 21) assessed its performance on HIV-2 subtype A samples only and claims detection limit of 200 c/ml. This is not what is stated here. I suggest to remove HIV-2 from this sentence.Also the limit of quantitation for HIV -1 has changed during the years. Please provide the version number of the VL kit used in this study and give the updated LOQ for the version used herein .LINE 151: Replace HIV by HIV-1 (to exclude HIV-2)Line 179: vl <40 cannot considered undetectable. If 40 is the quantification limit It could be considered as below quantification limit. If <40 is the LOQ for the assay used herein, please amend the term “undetectable” ( in cases of VL <40 C/ML) to “below quantification limit” all through this paper.Line 282: Fi 1 change to Figure 1Line 296: term “suppressed viral load” and “undetectable viral load” should be explained in the text and not only in the figure legend, especially as <1000 is not regularly considered “suppressed…”. Please discuss to these numbers in the discussion section as today VL >200 (SOMETIMES >50) is considered virological failure.Table 1 : need restructuring. Numbers are not in lanes and some do not fit the spaceTable 3: needed reformatting and detailing. What does NA mean? Why some spaces are empty? The list of ART for each sample is the drugs which could be taken and are no affected by the identified DRM? Why to include drugs that are not in use any more? The subtype is given for both RT and PR? Please provide detailed information so that the reader will easily understand.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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 #3: 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: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: 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 #3: 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 #3: Yes**********6. Review Comments to the AuthorPlease 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: Just one small correction to make: revised clean text, line 93 "HIV positive FSW not known to be on ART" is confusing, should be "HIV positive FSW not on ART"Reviewer #3: This is a strong paper that I enjoyed reading. The one remaining issue that I would strongly recommend the authors to consider is to present a comparison of baseline characteristics of participants who did and not complete the follow-up to give readers a better sense of the types of participants who dropped out of the study.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  9 Dec 2019Note from the Editor:1) Page 8 line 139: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT). In Table 1: N. gonorrhoeae or C. trachomatis. Please choose either NG/CT or N. gonorrhoeae - C. trachomatis throughout the text.Response: We have chosen to use N. gonorrhoeae and C. trachomatis throughout the text ; except for the name of the Probetec assay used to test for these infections, as NG/CT is part of the brand name of the assay. 2) Page 9 148-151: nuclisens can officially assess HIV-1 only (not HIV-2). Only one publication (ref 21) assessed its performance on HIV-2 subtype A samples only and claims detection limit of 200 c/ml. This is not what is stated here. I suggest to remove HIV-2 from this sentence.Response: Indeed, Biomerieux-diagnostics mentioned that NucliSens® HIV-1 QT assay is generally insensitive for detection of HIV-2 RNA. In addition, samples from individuals infected with HIV-2 may exhibit cross-reactivity in this assay. We therefore, have removed HIV-2 from this sentence, as suggested (page 9: line 151). Please also note that, as already stated in the manuscript, subjects with evidence of HIV-2 infection were excluded from the study.3) Also the limit of quantitation for HIV-1 has changed during the years. Please provide the version number of the VL kit used in this study and give the updated LOQ for the version used herein .Response: We used the NucliSens EasyQ® HIV-1 v2.0 kit. 48 tests - Ref: 28 5033. The kit is of high-level sensitivity with validated input volumes plasma, can detect the majority of HIV-1 subtypes including: A, B, C, D, F, G, H, J, CRF01_AE and CRF02_AG with a viral load measuring range between: 10-10 000 000 copies/ml. This kit provides detection limits according to volume of plasma used:Flexible Input Limit of Detection (LoD)1 ml plasma 25 cps/ml0.5ml plasma 50 cps/ml0.1 ml plasma 292 cps/mlDry Blood Spot (0.1 ml) 802 cps/mlWe used 0.5 ml plasma, therefore the lowest viral load we could amplify was 50 copies/ml (page 9: line 151). 4) Page 9 line 151: Replace HIV by HIV-1 (to exclude HIV-2)Response: We have replaced HIV by HIV-1 . 5) Page 10 line 179: vl <40 cannot considered undetectable. If 40 is the quantification limit it could be considered as below quantification limit. If <40 is the LOQ for the assay used herein, please amend the term “undetectable” (in cases of VL <40 C/ML) to “below quantification limit” all through this paper.Response: Thank you very much for underlining this ambiguous misuse of “undetectable” instead of “below quantification limit”, “undetectable” being a term too often used in viral quantification. Since we used the NucliSens EasyQ® HIV-1 v2.0 kit. 48 tests - Ref: 28 5033 and 0.5 ml plasma, the lowest viral load we could amplify was 50 copies/ml. We therefore have amended the term “undetectable”  to “below quantification limit” all through the manuscript, and the figure 2. Please, see .6) Page 18 line 282: Fi 1 change to Figure 1Response: We have changed Fi 1 to Figure 1 (page 18: line 290).7) Page 19 line 296: term “suppressed viral load” and “undetectable viral load” should be explained in the text and not only in the figure legend, especially as <1000 is not regularly considered “suppressed…” Please discuss to these numbers in the discussion section as today VL >200 (SOMETIMES >50) is considered virological failure.Response: The term viral suppression or “suppressed viral load”, when viral load is <1000 copies/mL was used to evaluate treatment success by the World Health Organisation (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS). According to the 2016 WHO Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection, a viral load <1000 copies/mL defines treatment success. The term “undetectable” viral load was defined as viral load below detection limits of the assay used for viral load quantification in the study . For the latter, we however now use “below quantification limit” instead of “undetectable” throughout the manuscript.8) Table 1: Needs restructuring. Numbers are not in lanes and some do not fit the spaceResponse: We have restructured Table 1 so that numbers are in lanes and fit the space (page 14-17).9) Table 3: needed reformatting and detailing. What does NA mean? Why some spaces are empty? The list of ART for each sample is the drugs which could be taken and are no affected by the identified DRM? Why to include drugs that are not in use any more? The subtype is given for both RT and PR? Please provide detailed information so that the reader will easily understand.Response table 3: NA meant not applicable as there was no resistance mutation to this class of ARVs; some spaces were empty for the same reason. We have now decided to leave all these spaces empty when no resistance mutation was present to a given ARV class. The list of specific drugs is quite useless at the end and we have suppressed this column entirely in order to make the table easier to read. We have provided the sequences corresponding to the protease and the reverse transcriptase regions (PR/RT), and this is now better explained (table page 23-24: table 3).AttachmentResponse to reviewers_ii.docxSubmitted filename: Click here for additional data file. 16 Dec 2019HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, BeninPONE-D-19-15725R2Dear Dr. Alary,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Orna MorAcademic EditorPLOS ONEAdditional Editor Comments :Thank you for addressing all issue and correcting the paper. I have enjoyed reading it and look forward to seeing it on pubmed.I still find Table 3 not lined up- but I guess you can improve it when the paper is processed for publication.Reviewers' comments: 8 Jan 2020PONE-D-19-15725R2 HIV treatment response among female sex workers participating in a treatment as prevention demonstration project in Cotonou, Benin Dear Dr. Alary:I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Orna Mor Academic EditorPLOS ONE


South Africa has the highest HIV prevalence and supports the largest antiretroviral therapy (ART) programme globally. With the introduction of a test and treat policy, ensuring long term optimal adherence to ART (≥95%) is essential for successful patient and public health outcomes. The aim of this study was to assess long-term ART adherence to inform best practices for chronic HIV care.p-values) was used to determine the effect of pill burden and concurrent ART and tuberculosis treatment on adherence.Long-term ART adherence was retrospectively analysed over a median duration of 5 years (interquartile range [IQR]: 5.3–6.5) in patients initially enrolled in a randomised controlled trial assessing tuberculosis and HIV treatment integration and subsequently followed post-trial in an observational cohort study in Durban, South Africa. The association between baseline patient characteristics and adherence over time was estimated using generalized estimating equations (GEE). Adherence was assessed using pharmacy pill counts conducted at each study visit and compared to 6 monthly viral load measurements. A Kaplan Meier survival analysis was used to estimate time to treatment failure. The McNemar test  and median time on ART was 70 months (IQR = 64–78). Mean adherence was ≥95% for each year on ART. Stable patients provided with an extended 3-month ART supply maintained adherence > 99%. At study end, 96 and 94% of patients were optimally adherent and virologically suppressed, respectively. Time since ART initiation, female gender and primary breadwinner status were significantly associated with ≥95% adherence to ART. The cumulative probability of treatment failure was 10.7% at 5 years after ART initiation. Concurrent ART and tuberculosis treatment, or switching to a second line ART regimen with higher pill burden, did not impair ART adherence.Optimal long-term adherence with successful treatment outcomes are possible within a structured ART programme with close adherence monitoring. This adherence support approach is relevant to a resource limited setting adopting a test and treat strategy. Currently, an estimated 36.7 million people are living with HIV (PLWH) worldwide . South AAccess to ART is only one aspect of an effective HIV management programme. Optimal adherence to ART is essential and early studies reported that ≥95% adherence to ART was required to achieve and maintain viral suppression , 7. AlthShort term ART adherence rates (less than 2 years from treatment initiation) in South African patients from multiple urban HIV clinics is reported to range from 63 to 88% –27, howeThe aim of this cohort study was to assess long-term adherence in a cohort of patients on ART for at least 5 years at the Centre for the AIDS Programme of Research in South Africa’s (CAPRISA) eThekwini Clinical Research site in Durban, South Africa.n = 642). Details of the study outcomes have been published elsewhere [Initially, HIV and tuberculosis (TB) co-infected patients were enrolled in the Starting Antiretroviral Therapy at Three Points in Tuberculosis (SAPiT) open label randomised controlled trial from June 2005 to July 2008  [After completion of follow up in the SAPiT trial, patients were offered enrolment into a prospective observational study, TB Recurrence upon Treatment with HAART (TRuTH), investigating the rate of TB recurrence in HIV infected adults on ART who had completed pulmonary TB treatment (n = 402) , 34. Virn = 402) . PatientAll patients received pre-ART education and ongoing adherence support counselling by trained counsellors and site pharmacists post-ART initiation. Pillboxes were provided to all patients as an adherence aid. Patients who did not come in on their scheduled clinic appointment date were telephonically or physically contacted. Close family members or friends were enlisted as treatment supporters, where possible, to provide additional social support for patients identified with adherence challenges.The SAPiT and TRuTH studies were conducted under the oversight of the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu-Natal . Participants in the SAPiT and TRuTH studies provided written informed consent. The current study is a retrospective secondary analysis of previously collected anonymized data from the parent trials with no direct participant contact (BREC ref.: BE046/16).To assess long-term adherence, we conducted a retrospective analysis in a sub-cohort of patients on ART for at least 5 years from the time of ART initiation in the SAPiT trial until completion of follow up in the TRuTH study. To maintain continuity of care, patients accessed ART via the CAPRISA AIDS Treatment (CAT) programme at the same clinic after completion of follow up in SAPiT and before enrolment in TRuTH. Patients who never initiated ART, were lost to follow up in the SAPiT trial, did not receive ART from the site’s research pharmacy and for whom pill count data was missing for more than 6 consecutive months in either study were excluded.Adherence to ART in both studies were determined from pharmacy pill count data and viral load measurements. Pill counts were conducted by the study pharmacist at monthly study visits in the SAPiT trial and at monthly or 3-monthly study visits in the TRuTH study. Adherence percentage was calculated using the following formula :\documeOptimal adherence was defined as ≥95% of doses taken in the time between the study visits . Pill coBaseline demographic data were analyzed by descriptive statistics and reported as median with inter-quartile range (IQR) or proportions. Undetectable viral load was defined as < 400 copies/mL as per the South African national treatment guidelines at the time. In the SAPiT trial, treatment failure was defined as two HIV-1 RNA measurements of > 1000 copies/mL taken at least 4 weeks apart followed by ART discontinuation or change of all drugs in the ARV regimen. In the TRuTH study treatment failure was defined as two consecutive HIV-1 RNA measurement of > 1000 copies/mL at least 6 months apart followed by a change to a second line boosted lopinavir/ritonavir (LPV/r) based regimen.p-value < 0.2 in the unadjusted analyses. Interaction terms were incorporated into the multivariable model, one at a time, and were retained if found to be significant. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)  were calculated to assess the extent to which mean pill count adherence (< 95% vs ≥95%) for each year on ART could be used to predict viral load outcomes (< 400 copies/mL vs ≥400 copies/mL) at the end of the same 12 month period. The McNemar test  was used to determine the impact of concurrent ART and TB treatment on adherence to ART by analyzing changes in ART adherence and viral load suppression rates before and during TB treatment. The McNemar test was also applied to the adherence proportions and viral load suppression rates before and after switching to a second line regimen to assess the impact of the pill burden on adherence. A Kaplan Meier survival analysis was used to estimate time to treatment failure. All statistical tests were conducted at 5% level of significance. Statistical Analysis System (SAS) version 9.4 software was used to conduct statistical analysis.Mean pill count adherence was calculated as the weighted mean of the monthly and/or 3-monthly adherence values over that time period, where the weights were calculated as the length of time between each study visit. The association between baseline patient characteristics and adherence over time was estimated using generalized estimating equations (GEE) with a logit-link function and exchangeable working correlation structure. A univariable analysis was first performed, and thereafter a multivariable model was estimated with gender, age and factors with a 3 (IQR:76–249) and median viral load was 141,000 copies/mL . Most patients (254/270) were classified as WHO stage 3 at baseline. A third of the cohort had completed secondary school, 60% were in full or part-time employment and more than half of all patients had disclosed their HIV status. The median duration on ART from initiation to completion of follow up was 70 months (IQR: 64–78).A total of 270 patients met the inclusion criteria for this analysis Fig. . The medThe time period between patient exit from SAPiT and enrolment in TRuTH (where no pill count data available) was a median of 13 months IQR: 5–21). The number of patients for whom pill count data was available in each 6 month period is shown in Table 1. The nun = 267) were provided with an extended 3-month ART supply at a total of 1061 study visits. Overall mean adherence for patients who received a 3-month ART package was 99.5%.In the 6 months post-ART initiation, 91.8% of patients had optimal adherence. Half of all patients had at least one sub-optimal adherence measurement (< 95%) in the first 6 months after starting ART, less than 20% between the first and sixth year and 23% after 6 years on ART  occurred within 2 years after ART initiation. The cumulative probability of treatment failure was 10.7% at 5 years after ART initiation  than those reported in several African studies, where adherence estimates ranged from 72 to 94% , 37–41. Female patients were 33% more likely to have optimal adherence compared to males in our cohort. Although gender has not been consistently found to influence ART adherence, HIV infected women in developing countries tend to demonstrate better health seeking behaviours than men which may be motivated by their role as the primary care giver in their families , 53. BeiViral suppression rates exceeded 90% for each year throughout the observation period. Similar long-term virologic outcomes have been reported in other low- and middle-income regions , 54–58. Sensitivity was high, but specificity was low for the use of pill count data as a proxy for viral load outcomes. Whilst some studies have demonstrated an association between pharmacy adherence measures and virologic outcomes others have found poor agreement between the two measures , 62–65. Patients who were provided with an extended 3-month ART supply in the TRuTH study were able to maintain high adherence levels over the extended time between clinic visits. This finding provides reassurance that stable patients can maintain optimal adherence when provided with an extended ART supply and patients who successfully adhere to treatment in the first 2 years are ideal candidates for more than 1 month’s supply of treatment. In an overburdened public health care setting, such as South Africa, this practice can ease clinic patient load and reduce the need for monthly clinic visits which impact negatively on patient finances and employment prospects.High pill burden, regimen complexity and dosing more than once a day are known to hinder adherence . DespiteHIV and TB co-infection rates are highest in South Africa . ConcurrThere are several limitations to our study. This was a retrospective study of ART adherence conducted at a single research site and adherence behaviour may have been influenced by participation in a clinical trial, where patients were routinely monitored and received extensive adherence support and counselling. Factors associated with adherence behaviour were limited to available baseline patient and clinical data. In addition, our patients were initiated on ART at lower CD4 counts with advanced HIV disease and sicker patients may be more motivated to adhere to treatment . Hence, Optimal long-term adherence rates observed in this cohort support the evidence of high adherence reported in other long-term African patient cohorts. Pill count should not be used as a standalone measure of adherence as it was not a good predictor of viral failure and viral load measurement remains the benchmark for monitoring treatment response. However, adherence during the 6 months post-ART initiation period has been shown to impact long-term treatment outcomes and, per in-country treatment guidelines, viral load is only measured 6 months after ART initiation and thereafter annually. Therefore, we recommend adherence monitoring using pill count as a quick, simple adherence measure at each visit in this critical 6 month period to identify patients with early adherence challenges and allow for timeous intervention.Provision of adherence support measures which may have assisted patient adherence included individual and group counselling, provision of pill boxes, treatment supporters and tracking of patients who missed their clinic appointment date. Pharmacists were also able to identify patients experiencing adherence challenges when conducting pill counts and could either provide or refer patients for appropriate adherence counselling. We also demonstrated that providing stable patients with a 3-month ART supply was feasible as optimal adherence was still maintained over this extended period. This has the potential to reduce clinic workloads and patient time and costs associated with monthly clinic visits and is currently being investigated further in a randomized control trial . SeveralIn summary, our study found optimal long-term adherence with good treatment outcomes in a structured ART programme with close adherence monitoring and support. With the implementation of a test and treat policy and rapid scale up of ART provision, it is imperative that patient adherence is monitored early on from ART initiation and then continually assessed and supported, to ensure a successful ART programme in South Africa.


Incomplete virologic suppression results in mutations associated with resistance and is a major obstacle to disease control. We analyzed the genotypic profiles of HIV-1 patients at the time of the first virologic failure and the response to a salvage regimen after 48 weeks.This work was a cross-sectional, retrospective, analytical study based on data collected from medical records and genotyping tests between 2006 and 2016. The sample consisted of data on individuals living with HIV (PLWH) from three major reference centers.A total of 184 patients were included in the data analysis. Viral subtype B was the most common (81.3%) as well as M184 V/I (85.3%) and K103 codon mutations (65.8%). Forty-eight weeks after switching to a salvage regimen, 67.3% of patients achieved an undetectable viral load.p = 0.179). An ARV time (the beginning of the first ARV regimen up to genotyping) of > 36 months was a protective factor for detectable viral load  and a risk factor for developing ≥3 NRTI(t)-associated mutations .The number of mutations associated with nucleos(t)ide reverse transcriptase inhibitors (NRTI(t)s) did not affect virologic suppression (9.3% for zero NRTI(t)-associated mutations vs 48.6% for 1–2 NRTI(t)-associated mutations vs 42.1% for ≥3 NRTI(t)-associated mutations, We found that extensive resistance to NRTI(t)s at the time of the first virologic failure did not impact virologic suppression at 48 weeks after switching to a second-line therapy based on NRTI(t)s plus protease inhibitors. Approximately 21 million people are living with HIV (PLHIV) and receiving antiretroviral therapy . HoweverIn Brazil, approximately 60% of PLHIV receive ARVs  through 10 on patients who failed first-line therapy between 2013 and 2015 found a higher prevalence of the M184 V/I mutation (74.3%) and K103 codon (56.7%), similar to that found in other Brazilian states [Genotypic and immunovirologic data from patients following the first virologic failure are scarce in Brazil, and they are limited to certain regions of the country. A recent study from the city of São Paulon states . Among tLow- and middle-income countries in sub-Saharan Africa, where 70% of PLHIV live , access Mutations are associated with high reduced susceptibility or virologic response to relevant NRTIs. Mutations reduce NRTI susceptibility or virologic response, which contributes to reducing susceptibility in combination with other NRTI-resistant mutations. The impact of mutations associated with NRTI(t)s during the second-line regimen was evaluated through the SECOND-LINE, EARNEST and SELECT studies –15 and tThis work was a cross-sectional, retrospective, multicenter study evaluating genotypic resistance tests of PLHIV during the first virologic failure in the city of Recife, the capital of the state of Pernambuco, an important medical center in Northeast Brazil. The patients were enrolled in three large centers specializing in the care of PLHIV. We reviewed tests that were performed from January 2006 to December 2016 and included patients over 18 years of age who had used ARVs for more than six months and presented two consecutive viral load measurements, with an interval of at least 30 days, and values greater than 1000 copies/ml. Individuals who underwent ARV switching due to virologic failure without genotyping tests, those who used ARVs before receiving the first-line therapy, those showing an absence of mutations associated with resistance in genotyping, and those with insufficient data were excluded.The immunovirologic and genotypic data and the history of ARV use were collected from genotyping tests, medical charts and through the Laboratory Test Control System (Sistema de Controle de Exames Laboratoriais - SISCEL- online platform). To identify the mutations associated with resistance, the ViroSeqTM HIV-1 Genotyping System kit  was used from 2006 to 2008 and the TRUGENE System  was used from 2009 to 2016 by the laboratories that served the National Genotyping Network . The viral load (VL) was measured until July 2013 using the Versant HIV-1 RNA 3.0 assay ; real-time qPCR was used after this date . The CD4 T-cell counts (CD4) were performed by flow cytometry .https://hivdb.stanford.edu/hivdb/by-mutations) was used to interpret the genotype resistance. The mutations were analyzed individually using a list provided by the International AIDS Society (IAS) in 2017, [The Stanford HIVdb program version 8.4  was calculated individually only for the drugs used by each patient in the salvage regimen. A value of 1 was attributed to drugs with full activity, 0.75 for a low potential resistance level, 0.5 for a resistance level, 0.25 for an intermediate resistance level and zero if the drug showed a lack of activity.SPSS 13.0  for Windows and Excel 2010 were used for the statistical analysis. The results are presented in the tables with their respective absolute and relative frequencies. The mean or median values for the quantitative variables were calculated according to the normality of their distribution. The normality of the quantitative variables was tested using the Kolmogorov-Smirnov test.p < 0.05 was considered statistically significant. Poison-regression was chosen as excellent alternative to estimate the adjusted prevalence ratio for confounding variables in cross-sectional studies compared to Cox and log-binominal logistic regression. The study was approved by the institutional ethics committee (CEP-CCS-UFPE/1.985.922).We tested for the presence of an association using the chi-square test and Fisher’s exact test for categorical variables. To identify the predictive factors that independently influenced virologic suppression and the number of mutations associated with NRTI(t)s, we used the STATA/SE 12.0 programs. For the multivariate analysis, the Poisson regression model was used, taking into account the variables that obtained significance ≤0.20 in the bivariate analysis. The prevalence ratio was calculated with a 95% confidence interval. A A total of 534 medical records were evaluated, of which 270 were excluded due to a lack of genotyping tests when patients switched ARVs after the first virologic failure. In 59 medical records, there was no selective pressure during genotyping. Therefore, the absence of mutation does not represent reality. Thus, 59 patients were excluded. Non-adherence to ARV use at the time of genotyping was recorded in the medical charts of the 59 excluded patients with absence of resistance-associated mutations. Furthermore, 21 patients were not either illegible or had insufficient data.n = 155) and VL (n = 141) pre-ARV were 176 cells/m3 and 130,000 copies/ml, respectively.A total of 184 patients were included in the first virologic failure for analysis. The majority of the participants were male (73.4%) and the mean age at virologic failure was 41 (±9.4) years. The median CD4 . Most individuals (89.7%) exhibited at least one mutation for NRTI(t). The association of the presence of ≥3 NRTI(t)-associated mutations with CD4 pre-ARVs < 200 cell/mm3 , with CD4 at the time of genotyping < 200 cells/mm3 , and with a time on ARV of greater than 36 months  were significant, according to Table The genotypic, immunovirologic and ARV characteristics at the time of genotyping are described in Table p = 0.002) and who developed VL at the time of genotyping of 10,000–100,000 copies/ml  and > 100,000 copies/ml  had a higher risk of having ≥3 NRTI (t)-associated mutations.After analysis using the Poisson regression model Table , only pap = 0.051). K65R emerged in 7.6% of 184 genotyping tests  of the genotyping tests. Among those who used NNRTI, this mutation occurred in 78.0% (n = 121/155). The NNRTI-associated mutation 90IV was more frequent in the non-B subtype  and the 190AS was most prevalent for etravirine.There was a trend towards the emergence of three or more thymidine-associated mutations (TAMs) when the time on ARVs was greater than 36 months  were on lopinavir/ritonavir (LPV/r) and 41.4% (n = 12) were on atazanavir (ATV) , as did the PI-associated accessory mutations 10IFV  and 20RT .The PI-associated major mutation 54VL emerged frequently in subtype B sequences .The total number of mutations was significantly equal to or greater than seven among the users who had a CD4 count below 200 cells/m3 at the time of genotyping  patients after the genotyping test was performed. Thus, 176 (95.7%) patients switched to the salvage regimen. Of these individuals, 109 (61.9%) patients used TDF/3TC as the NRTI(t), followed by 28 (15.9%) who used TDF/AZT(zidovudine)/3TC and 27 (15.3%) AZT/3TC. Only nine (5.1%) individuals did not have NRTI(t)s included in their salvage regimen.n = 32). When the salvage regimen consisted of two active drugs (GSS = 2), 79.5% of this population showed genotyping with less than three NRTI(t)-associated mutations (79.5% vs 20.5% for ≥3 NRTI(t)-associated mutations, p < 0.000).PIs were present in the salvage regimen of 99.4% of the individuals; only one patient did not use PI. LPV/r was the most frequently prescribed (48.5%) followed by ATV/r (33.1%). Integrase inhibitors made up the salvage regimen of 35 (19.9%) individuals. Raltegravir (RAL) was the most frequently used one (The susceptibility of etravirine was not associated with the use of EFV or nevirapine (NVP) or with the number of NNRTI-associated mutations. This treatment remained fully active in 63.2% of the sequences among the patients who experienced a failed NNRTI therapy. We found a similar susceptibility of AZT and TDF (64.2% vs 65.9%). Among the PI/r users, darunavir/ritonavir (DRV/r) showed the highest percentage of sensitivity (69%), and ATV/r experienced the highest percentage of resistance (79.3%).p = 0.013) and on ARV for more than 36 months .Most of the salvage regimens showed GSS equal to two (42.6%) or greater than two (44.3%). We found that in most of the regimens with a GSS greater than 2, the patient was in virologic failure for more than 24 months . The significant majority of TDF users in virologic failure were salvaged with an ARV regimen with a GSS equal to two .When virologic failure occurred during LPV/r use, the GSS of the salvage therapy regimen was significantly higher than two  experienced virologic suppression and 11.3% did not reach an undetectable VL but had below 400 copies/ml. The majority of the population with undetectable VL had more than 36 months of ARV use until genotyping , and 42.0% were in virologic failure for 12 to 24 months  . Of these, 67.3% (p = 0.179) (Table The number of NRTI(t)-associated mutations did not affect virologic suppression (9.3% for zero NRTI(t)-associated mutations vs 48.6% for 1–2 NRTI(t)-associated mutations vs 42.1% for ≥3 NRTI(t)-associated mutations, 9) Table .Table 5GAfter an analysis with the Poisson regression model, only being on ARVs for more than 36 months until genotyping was a protective factor for a detectable viral load  48 weeks after switching to the salvage regimen (Table p = 0.047) and when the CD4 at the time of genotyping was below 200 cells/mm3 (81.4% vs 18.6% for CD4 < 100 cells/mm3 p = 0.010).For the 153 patients with documented CD4 after 48 weeks, the median was 376 cells/mm3  and the median CD4 gain was 125 cells/mm3 . In the population with virologic success, the variation in the CD4 gain above 100 cells/mm3 was significant when the VL at the time of genotyping was 10,000–100,000 copies/ml  and NNRTI-associated mutations, with no impact on virologic suppression. We observed that the salvage therapy regimen was predominantly composed of PI/r and NRTI(t)s, with virologic success in most cases. Subtype B remains the most common in Pernambuco –19 and iThe elevated presence of M184 codon mutations is expected and arises as a consequence of the use of lamivudine as part of all the first-line regimens in our study. This drug confers a high level of resistance to cytosine analogs (lamivudine and emtricitabine), a low level of resistance to abacavir, and the increased susceptibility of zidovudine and TDF. In addition, it decreases the replication capacity of HIV-1 , 24. ItsSimilar to our results, the high prevalence of M184 V/I mutations was reported in several regions of Brazil , 25, 40,10 as well as with the C subtype [We found no association between the number of NRTI(t)-associated mutations and the ARVs used at the time of genotyping, including ARV regimens with or without TDF. There are studies showing a greater number of resistance-associated mutations among AZT  and TDF  subtype . The assThe K103 N/S mutation was documented in most genotypic sequences, due to the frequent use of EFV as the primary NNRTI in the first-line therapy for more than a decade in Brazil. These findings are similar to the national data  and dataThis analysis was hampered by the small number of NVP users at the time of virologic failure. However, some studies –34 have The median time from ARV therapy to genotyping was similar to that of recent studies in Brazil , 10 AfriA shorter time on ARV is used as the best scenario for adherence in the calculation of odds ratio in studies that seek to understand the risk factors for treatment adherence. However, after association testing, its impact on adherence is not always confirmed . This fiBy contrast, more than 36 months on ARV increased the risk for the selection of three or more NRTI(t)-associated mutations, although a longer time in virologic failure did not generate the same outcome. According to the virologic failure criterion of this study, individuals with low VL (> <1000 copies/ml) were not considered to be in virologic failure, because the genotyping tests available in Brazil during the data collection period were not able to expand the genetic material when the VL was < 1000 copies/ml. However, there is evidence that having a VL of > 200 copies/ml increases the risk for virologic failure and may lead to mutation accumulation . TherefoWe observed that the presence of NRTI(t)-associated mutations or TAMs at the first virologic failure did not interfere with virologic suppression 48 weeks after the switch to a second-line therapy with NRTI(t)s combined with PI/r. This finding is not only due to the high potency of PI/r or to the direct activity of NRTI(t)s, and it may reflect an effect of the NRTI(t)s on viral fitness . This fiWe found no difference in the virologic response between regimens with an individual GSS of less than two and those greater than or equal to two, confirming the hypothesis that there is no difference between active, partially active or inactive NRTI(t)s in terms of virologic success . In the It is possible that pharmacokinetic characteristics optimize the benefits of NRTI(t)s even if inactive. Thus, ARVs with a long intracellular half-life, such as TDF and lamivudine, may help the PI/r to maintain viral suppression. This effect is independent of the drug activity .We were able to define the time in virologic failure until the switch to second-line therapy. Surprisingly, most of the patients with undetected viral loads were in virologic failure for more than 12 months. Because the time under virologic failure did not interfere with the number of NRTI(t)-associated mutations or TAMs in our study, we can suggest that the resistance-associated mutations arose during the first months of virologic failure. In the EUROSIDA study , a high The total absence of ARV selective pressure during part of the virologic failure period is another way to explain why the longer virologic failure time did not influence the number of NRTI(t)-associated mutations or TAMs. The complete withdrawal of ARVs generates fewer resistance-associated mutations than does the maintenance of sub-therapeutic doses . It was When we analyzed the virologic response and ARVs used in salvage therapy, we observed that there was no difference between the patients who used AZT or TDF or both. The use of LPV/r was significant among those who experienced virologic success, probably because it was the most frequently prescribed PI during the study period, as directed by the Brazilian Ministry of Health. Among users of DRV/r or RAL, the majority achieved virologic success, although they were smaller in absolute numbers.DRV/r is the newest PI and has a high genetic barrier, in addition to fewer adverse effects than LPV/r, possessing activity even with a protease mutation . When thOur primary limitation was the difficulty in establishing cause-and-effect relationships, because this was a cross-sectional study. Our results may have been partially affected by data we were unable to collect, particularly those related to adherence to ARV therapy, and by the loss of information in some variables. However, this study has relevant points because it updates the genotypic data of an important region of Brazil with limited resources. In addition, we were able to establish the time in virologic failure and the ARV regimen before and after genotyping in the first virologic failure as well as the factors associated with the number of NRTI(t)-associated mutations and virologic success.Patients in their first virologic failure who were seen at referral centers in the city of Recife, Pernambuco, Northeastern Brazil presented a high frequency of mutations pertaining to secondary resistance to the use of NRTI(t)s and NNRTIs. However, extensive resistance in reverse transcriptase did not impact the second-line virologic suppression after 48 weeks of the salvage therapy with PI/r combined with active, partially active or inactive NRTI(t)s. The time on ARVs to genotyping was an independent protective factor for a detectable viral load. Long-term follow-up is needed to support the use of partially active NRTI(t)s in salvage therapy regimens while controlling for adherence-related factors. Studies assessing the actual need for genotyping after the first virologic failure with NNRTIs are also needed.