Healthcare services may consider the evidence provided by the present study as an opportunity to include HIV patients in a more adequate antiretroviral treatment arm, guaranteeing a personalized clinical pathway, thus becoming more efficient and effective over time.The aim of this study is to analyze the potential advantages of emtricitabine/tenofovir alafenamide (FTC/TAF) introduction, creating evidence-based information to orient strategies to reduce costs, thus preserving effectiveness and appropriateness. An Health Technology Assessment (HTA) was implemented in the years 2017–2018 comparing the dual backbones available in the Italian market: FTC/TAF, FTC/TDF (tenofovir disoproxil fumarate/emtricitabine) and ABC/3TC (abacavir/lamivudine). From an efficacy point of view, FTC/TAF ensured a higher percentage of virologic control and a better safety impact than FTC/TDF . From an economic point of view, the results revealed a 4% cost saving for the Italian National Healthcare Service NHS with FTC/TAF introduction compared with the baseline scenario. Qualitative perceptions’ results showed that FTC/TAF would decrease the burden of adverse events management, increasing the accessibility of patients to healthcare providers (FTC/TAF: 0.95, FTC/TDF: 0.10, ABC/3TC: 0.28;  The discovery of Highly Active Antiretroviral Therapies (HAART) and their introduction into clinical practice has transformed the human immunodeficiency virus (HIV) infection from a fatal condition to a chronic disease, decreasing morbidity and mortality as well as increasing life expectancy . HAART hIn the HIV setting, innovation is currently playing a key role since treatment choices and management practices should ensure patients’ long-term health with minimal comorbidity . This asIn this regard, the introduction of emtricitabine/tenofovir alafenamide (FTC/TAF) and the change of price setting of tenofovir disoproxil fumarate/emtricitabine (FTC/TDF) in Italy in 2017 has altered the HIV drug market which had been represented by triple therapy with the administration of FTC/TDF and abacavir/lamivudine (ABC/3TC). The rationale behind the development of TAF was to produce a therapeutic moiety with the potency of TDF combined with an improved safety profile . While TAs a consequence, this has had an impact on policymaking processes, disinvestment strategies and allocation of economic resources. An information gap has emerged concerning the management of HIV patients. While HIV is a significant economic burden for healthcare providers, limited country-oriented evidence is available with regard to the resource absorption  or multiMoving on from these premises, the aim of the proposed study is to analyze the potential advantages of FTC/TAF introduction creating evidence-based information to orient strategies useful to reduce costs in a context of budget cuts, also preserving effectiveness and appropriateness and guaranteeing universality and equity of care for HIV treatments, using a multi-dimensional approach  evaluation).To achieve the abovementioned study objective, a complete HTA was implemented. The HTA approach aims at providing decision makers with relevant and reliable information in selecting technologies with the best value to cost ratio, considering evidence-based and multi-disciplinary methodologies to evaluate processes and a number of criteria that deal with their economic, social, clinical, ethical and organizational implications ,11,12,13The introduction of healthcare technologies requires a deep analysis of direct expenditures and more attention to the related assessment, selecting only the most promising healthcare technologies, avoiding waste on innovations. HTA could support decision-making processes both at institutional–macro level and hospital–meso level. In the proposed analysis, the institutional National Healthcare Service perspective was taken into account.The HTA strategy is a useful method to support the decision-making process, particularly considering investment or disinvestment. At the same time, HTA could support lean thinking and lean management, suggesting the area of optimization and improvement .This HTA study was conducted in the years 2017–2018, comparing the dual NRTI backbones available in Italy: FTC/TAF, FTC/TDF and ABC/3TC, administered to both naïve and experienced HIV populations.Due to the multi-dimensional nature of HTA, several aspects of these drugs were analyzed (using a comparative approach), as described in the EUnetHTA Core Model : (i) genThe above dimensions were deployed, considering a literature review, economic and budget impact analyses  and qualBefore starting the assessment of the dimensions, the Problem/Population, Intervention, Comparator and Outcomes (PICO) approach  for the The evidence in the literature came from a systematic search of databases  from January 2011, to June 2018. The search terms included the following: “HIV treatment”, “emtricitabine/tenofovir alafenamide”, “disoproxil fumarate/emtricitabine”, “abacavir/lamivudine”, “clinical effectiveness”, “virologic control”, “drug-related adverse event”, “HIV naïve patients”, and “HIV experienced patients”. In accordance with the use of Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) [Peer-reviewed papers that explicitly described the clinical effectiveness and the safety profiles of TAF/FTC, TDF/FTC and 3TC/ABC were consequently included, and their quality was verified. The validation of the scientific evidence available on the topic was carried out by five experts , using the approach suggested by both the Critical Appraisal Skills Programme (CASP) checklist  for RCTsFor each selected study, the experts were asked to attribute first a qualitative synthesis of the results, and then, a score ranging from 4 (excellent) to 1 (insufficient) for the following items: (i) overall quality of the publication; (ii) generalizability of the results; (iii) completeness of the results.The evidence in the literature, in terms of the occurrence rate of drug-related adverse events and of data regarding virologic control, was used for the presentation of both the safety and the efficacy dimensions related to EUnetHTA Core Model approach.To adequately assess the economic dimension, it was necessary, firstly, to analyze and determine the HIV clinical pathway cost as the average process costs absorption; and secondly, to verify the affordability of the economic results, developing a budget impact analysis .Drugs’ costs (both HAART and other drugs): these were derived from the Italian ex-factory price updated to April 2018 and officially published on the NHS price list, considering the mandatory discounts required by law, and the VAT. For 3TC/ABC and TDF/FTC, the price of the generic drug published in the Italian Gazzetta Ufficiale  was conNon-drug costs: other medical costs for HIV+ patients management, including the total amount of hematologic, cultural and laboratory tests, diagnostic procedures, outpatient and specialistic visits, medical examinations, and hospital admissions. The clinical pathway related to the abovementioned procedures administered to patients was defined according to Delphi methods , involviCost of side effect management: the clinical pathway on an annual basis for patients who develop specific adverse events was evaluated , as already described, by means of Delphi approach , thanks The clinical pathway was assessed  considerThe economic analysis was performed with a time horizon of 12 months, assuming the National Healthcare Service perspective (third payer), according to the 2018 Italian Reimbursement Tariffs, for outpatients and hospital admissions, with a consistent approach with economic literature on the topic .The economic evaluation of the HIV patient clinical pathway was analyzed on the basis of both treatment-naïve and treatment-experienced populations, hence also differentiated considering the viral control achieved or not by the patients  of ≤50 or >50 copies/mL at 48 weeks), as already reported in the economic literature on the field .The process analysis of the HIV+ patients’ clinical pathways (stratified for populations characteristics) was conducted, and used also to perform a Budget Impact Analysis (BIA) using the above cost data inputs and showed the HIV healthcare evolution, up to three years. The BIA compared a baseline scenario, in which all eligible patients were treated with FTC-/TDF- and 3TC-/ABC-based regimens, with two different innovative scenarios .The first innovative scenario considered the introduction of FTC-/TAF-based regimens with the consequent complete disinvestment of FTC-/TDF-based regimens . The seMarket share considered for the development of the different scenarios derived from the Lombardy Region Clinical Pathway (CP), valid for the 2018, verified using the expert opinion of the Infectious Disease Department .After defining the comparative scenarios, the number of eligible patients was determined based on the Italian epidemiological data , and epidemiological estimates regarding HIV . In the Qualitative questionnaires derived from the issues proposed by EUnetHTA Core Model  were cirValidated questionnaires were used to examine the qualitative EUnetHTA Core Model dimensions. The perceived impacts of the three therapeutic strategies were assessed in terms of (i) equity and accessibility aspects, (ii) social determinants, considering the patients’ perspective, (iii) legal impact and (iv) organizational factors, considering a comparative approach. The questions were phrased as statements: the healthcare professionals answered considering the level of agreement and accordance, with a 7-item Likert scale ranging from −3 to +3 (where −3 = absolutely negative/no accordance and +3 = absolutely positive/complete agreement), considering all the possible multi-dimensional impacts of the alternative drugs on the patients’ pathway. Data were analyzed considering descriptive statistics. One-way ANOVA tests were used to describe the existence of statistically significant differences between the three studied backbones. All analyses were conducted with a significant level of 0.05. All the analyses were performed using the statistical software SPSS 22.0 and MS Excel.Out of the 1092 papers identified through database searching, only nine ,36,37,38Results deriving from the validation of literature were reported in The literature was used for efficacy and safety data retrieval . From anThe total cost of HIV+ patients management in one year, assuming the dual NRTI backbones, was evaluated  considerIn case of the change of price setting for FTC/TDF , the total cost for a naïve patient was EUR 7690.37 in case of VL ≤ 50 copies/mL and EUR 8313.53 in case of VL > 50 copies/mL, whereas the economic evaluation for a treatment-experienced patient was equal to EUR 7867.50 in case of VL ≤ 50 copies/mL and EUR 8519.84 in case of VL > 50 copies/mL.However, the introduction of FTC/TAF would require some additional investment in training courses  and meetings , for a medium-sized hospital, with an Infectious Diseases Department.p-value: 0.016), with significant improvement in equity profile. Moreover, this fact implied also a freeing up of human resources (approximately at least 80 min per patients per year) due to the reduction of healthcare services for the monitoring of renal and bone profiles ; this time could be invested in the care of other HIV+ patients.Focusing on the qualitative aspects , based op-value < 0.001), and patient quality of life , caused by long-term toxicities, considering patients’ perceptions of treatment outcome satisfaction.From a social perspective, FTC/TDF has a perceived negative impact on social costs , due to medicolegal issues in the prescription phase.With regard to the legal aspects, it should be noted that all the dual NRTI backbones in the study are mentioned in the Italian HIV National Guidelines . Expertsp-value < 0.001).From an organizational perspective, FTC/TAF could simplify the overall management of HIV+ populations’ drug complications for hospitals (0.63 versus −0.14 (FTC/TDF) and 0.006 (ABC/3TC), The HTA analysis provides clinicians, policymakers, and practitioners with evidence-based information useful to support HAART clinical choices in a general context of dynamic changing options for HIV treatment. Since HIV is a lifelong condition, its treatment needs to increasingly focus on the long-term management of patient complications and limiting treatment adverse effects, thus, also proposing simple dosages to reach a virologic control condition.Clinical trials ,34,35,36Despite regimens based on FTC/TAF or FTC/TDF seeming approximately equivalent in effectiveness (in terms of achievement of adequate virologic control), it should be noted here that FTC/TAF could broaden the number of eligible HIV patients, particularly those at high risk of developing renal and bone disease, as well as adolescents who have not yet reached peak bone mass.From an economic point of view, the results of the proposed study reported that the introduction of FTC/TAF could provide savings for the Italian NHS assuming a three-year time horizon. The lower medical costs associated with FTC-/TAF-based regimens reflect the reduction in adverse events management costs, with a higher cost decrease in treatment-experienced patients, compared with treatment-naïve patients, due to a higher development of serious adverse events.Economic advantage related to adopting FTC-/TAF-based regimens would range from a minimum of 4% to a maximum of 8%, depending on the possibility to administer the FTC/TDF as a generic drug (even if this option would present significant drawbacks due to long-term onset of drug-related long-term toxicities).The results of the study are consistent with scientific evidence available on the economic topic. An economic evaluation showed the economic superiority of FTC-/TAF-based regimens compared with FTC-/TDF- and ABC-/3TC-based regimens . Additiop-value = 0.000). Furthermore, the introduction of FTC/TAF would also generate organizational savings due to the reduction in the adverse events control activities, with the consequent freeing up of resources in terms of visits, laboratory tests, and overall management of complications. This positive organizational result could also consolidate the accessibility and equity profile of HIV+ treatments guaranteeing better care for the patients. This suggestion is also supported by the clinicians’ and patients’ perceptions, which are positive and incremental for FTC/TAF regimens .Focusing on the other HTA dimensions, FTC/TAF would be the preferable therapeutic strategy depending on the patient’s perception of improved quality of life, and, in general, considering the perceived social impact (The introduction of FTC/TAF was found to deliver improvements in efficacy, with a greater chance of virologic control and safety compared with the other dual NRTI backbones, FTC/TDF and ABC/3TC. An FTC/TDF disinvestment strategy would be a cost-saving option for the Italian NHS.To the best of authors’ knowledge, this study is the first attempt to fully evaluate the potential advantages of FTC/TAF introduction in clinical practice, with a multidimensional approach such as HTA methodology, offering new insights to advance the ongoing debate regarding the dual NRTI backbones given the paucity of economic resources.The analysis, albeit conducted using real-world data and perceptions of the specific Italian setting, may have important implications for policymakers, since cost reduction is always priority, particularly in the current era of economic recession. Strategies are needed to reduce costs, preserving effectiveness and appropriateness, and guaranteeing universality and equity of care. Healthcare services may consider the evidence provided by the present study as an opportunity to include HIV+ subjects in the most adequate HAART treatment arm, guaranteeing a personalized clinical pathway, thus becoming more efficient and effective over time.


Aiming to reach UNAIDS 90‐90‐90 targets, nearly all sub‐Saharan African countries have expanded antiretroviral therapy (ART) to all people living with HIV (PLWH) (Treat All). Few published data exist on viral load testing and viral suppression under Treat All in this region. We assessed proportions of patients with available viral load test results and who were virally suppressed, as well as factors associated with viral suppression, among PLWH in 10 Rwandan health centres after Treat All implementation.Cross‐sectional study during 2018 of adults (≥15 years) engaged in HIV care at 10 Rwandan health centres. Outcomes were being on ART , retained on ART , available viral load test results  and virally suppressed . We used modified Poisson regression models accounting for clustering by health centre to determine factors associated with being virally suppressed.3 . There was no statistically significant difference in viral suppression among patients who entered after Treat All implementation compared to those who enrolled before 2010 .Of 12,238 patients, 7050 (58%) were female and 1028 (8%) were aged 15 to 24 years. Nearly all patients  were on ART, of whom 11,198 (94%) were retained on ART. Among patients retained on ART, 10,200 (91%) had available viral load results; of these 9331 (91%) were virally suppressed. Viral suppression was less likely among patients aged 15 to 24 compared to >49 years : 0.83, 95% CI 0.76 to 0.90 and those with pre‐ART CD4 counts of <200 compared to ≥500 cells/mmIn this large cohort of Rwandan PLWH receiving HIV care after Treat All implementation, patients in study health centres have surpassed the third UNAIDS 90‐90‐90 target. To ensure all PLWH fully benefit from ART, additional efforts should focus on improving ART adherence among younger persons. To reacViral load monitoring is essential to ensure appropriate clinical decision making for PLWH, identify groups at risk of poor clinical outcomes and determine progress towards the 90‐90‐90 goals. Accordingly, the WHO recommends routine viral load testing at six months after ART initiation and at least annually thereafter . ReducedRwanda, a small East African nation with a population of nearly 13 million, became one of the first SSA countries to implement Treat All nationally in 2016. To date, few data on availability of routine viral load testing in Rwanda have been published, and none after Treat All implementation. Using routinely collected data from ten health centres, we aimed to describe prevalence of viral load testing and viral suppression over a 12‐month interval after a period of ART expansion and viral load scale‐up in Rwanda.22.1www.ca‐iedea.org). All research was performed according to the principles of the Helsinki Declaration and was approved by the Albert Einstein College of Medicine Institutional Review Board and the Rwanda National Ethics Committee, both of which waived patient consent because data were de‐identified prior to extraction into the research database.We conducted a cross‐sectional analysis of routine clinical data collected from January to December 2018 from an open observational cohort of patients receiving HIV care at ten Rwandan health centres affiliated with the Central Africa International epidemiology Databases to Evaluate AIDS  were enrolled in care as of 1 January 2018; (2) had ≥1 health centre visit after enrolment (and could therefore be considered to have engaged in care); (2) had ≥1 health centre visit during 2018; and (3) were not known to have died or transferred out during 2018. Visits included clinical appointments, pharmacy pick‐ups, or laboratory encounters. Each participating health centre routinely collects demographic, clinical and laboratory data as part of clinical care using standardized paper forms; these data are regularly entered into electronic databases and periodically extracted into the CA‐IeDEA research database after de‐identification. Data for this analysis were extracted into the study database on 25 September 2019.2.32) and WHO clinical stage .We defined the following outcomes: on ART , retained on ART , available viral load test results  and virally suppressed . Demogr2.4p < 0.05.We calculated proportions of patients on ART, retained on ART (among those on ART), who had available viral load test results (among those retained on ART), and who were virally suppressed . We then examined factors associated with viral suppression using modified Poisson regression models with robust variances to calculate crude and adjusted prevalence ratios (aPRs) and confidence intervals (CIs), with generalized estimating equations to account for clustering within health centres. Multivariable models were adjusted for all patient characteristics. Data were analysed using SAS 9.4 ; statistical significance for all tests was two‐sided at 33 (interquartile range: 240 to 543). In total, 11,933 patients (97%) were on ART, of whom 11,198 (94%) were retained on ART and 10,200 (91% of those retained on ART) had available viral load results  did not return for a visit in 2018 and were excluded from further analysis. Among the 12,238 patients in HIV care during 2018, 7050 (58%) were female and 1028 (8%) were aged 15 to 24 years. Most patients (97%) entered directly into routine HIV care and treatment programmes; 248 (2%) and 115 (1%) initially entered care into PMTCT or tuberculosis clinics respectively. Of all patients, 1596 (13%) entered care after implementation of Treat All in July 2016. Median pre‐ART CD4 count was 358 cells/mmlt Table .3 compared to ≥500  were virally suppressed. Viral suppression rates in the 10 study health centres ranged from 88% to 94% among patients with available results Table . Viral sAmong 1596 patients who entered care after implementation of Treat All, 1484 (93%) were on ART and 1323 of these (89%) were retained on ART. Of those retained on ART, 1174 (89%) had available 2018 viral load results, of whom 1036 (88%) were virally suppressed. In adjusted analyses, there was no statistically significant difference in viral suppression among patients who entered after Treat All implementation compared to those who enrolled before 2010 .4In this study of routinely collected data from HIV programmes in 10 Rwandan health centres during 2018, we found that very high proportions of active patients were on ART, had routine viral load testing performed, and were virally suppressed. These results provide early evidence of the successful scale‐up of both ART and viral load monitoring in a routine clinical setting after Treat All, suggesting that the UNAIDS 90‐90‐90 targets are attainable.We found high levels of viral suppression among patients whose viral load was measured. This is similar to data published from large controlled trials of universal test and treat in SSA , 13, 14 We found that viral suppression was less likely among patients aged 15 to 24 compared to older patients, results similar to those from multiple other studies in SSA Africa – including HTPN 071  and otheThe few studies describing rates of routine viral load testing in SSA indicate that coverage is variable , 9, 10. Like any investigation, this study was limited by certain factors. Our use of routine clinical data from patients engaged in HIV care did not allow us to estimate the proportion of PLWH with known HIV status, nor the proportion of those on ART among all PLWH with known status. Because the study focused on viral load testing and suppression, we limited the study to persons who were eligible for testing in 2018 (i.e. those in care in 2018), and thus did not account for patients who may have been lost to care earlier. We report the proportion of those virally suppressed among those with available viral load results, rather than among all those on ART, which could potentially have overestimated the rate of viral suppression. However, the minimal differences between study patients with and without available viral loads, as well similar rates of viral suppression observed in Rwanda’s PHIA  suggest 5In conclusion, we observed high rates of ART use, viral load monitoring and viral suppression among >12,000 PLWH actively engaged in HIV care at 10 Rwandan health centres. These are among the first published routine clinical HIV data after Treat All implementation in SSA and suggest that this policy can effectively contribute to attaining global HIV viral suppression targets.All authors have no conflicts of interest to disclose.JR, DH, QS and KA contributed to the study design, data analysis and interpretation. GM, MY and DN contributed to the study design and interpretation. AM, MR, ER, SN participated in data collection and interpretation. JR drafted the first version of the manuscript, which all authors subsequently reviewed, edited and approved.Table S1. Characteristics of patients retained on antiretroviral therapy, by availability of viral load – 10 health centers, Rwanda 2018 Table S2. Proportion of patients virally suppressed among those with available viral load, by site – 10 health centers, Rwanda 2018Click here for additional data file.


Understanding factors driving virological failure, including the contribution of HIV drug resistance mutations (DRM), is critical to ensuring HIV treatment remains effective. We examine the contribution of drug resistance mutations for low viral suppression in HIV-positive participants in a population-based sero-prevalence survey in rural South Africa.We conducted HIV drug resistance genotyping and ART analyte testing on dried blood spots (DBS) from HIV-positive adults participating in a 2014 survey in North West Province. Among those with virologic failure (> 5000 copies/mL), we describe frequency of DRM to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI), report association of resistance with antiretroviral therapy (ART) status, and assess resistance to first and second line therapy. Analyses are weighted to account for sampling design.Overall 170 DBS samples were assayed for viral load and ART analytes; 78.4% of men and 50.0% of women had evidence of virologic failure and were assessed for drug resistance, with successful sequencing of 76/107 samples. We found ≥1 DRM in 22% of participants; 47% were from samples with detectable analyte . Of those with DRM and detectable analyte, 60% showed high–level resistance and reduced predicted virologic response to ≥1 NRTI/NNRTI typically used in first and second-line regimens.DRM and predicted reduced susceptibility to first and second-line regimens were common among adults with ART exposure in a rural South African population-based sample. Results underscore the importance of ongoing virologic monitoring, regimen optimization and adherence counseling to optimize durable virologic suppression. The UNAIDS HIV epidemic targets for detection, sustained antiretroviral therapy (ART), and viral suppression propose that overall community viral suppression should reach 73% by 2020 . With teAmong patients in clinical research cohorts in sub-Saharan Africa with access to virologic monitoring, viral suppression at 12 months has been estimated between 84 and 90% among those on ART –13. In SWhile known factors contribute to virological failure , it remains unknown what proportion of those undergoing virological failure can be attributed to each factor. Studies in sub-Saharan Africa have found drug resistance mutations in 6–14% of ART-naïve patients , 19, 20,We conducted a population-based bio-behavioral survey in 2014 to characterize the HIV care continuum in a rural district of North West Province, an area of the country with substantial burden of disease and little available data . We noteData were collected from January–March 2014 in Lekwa-Teemane and Greater Taung sub-districts, within Dr. Ruth Segomotsi Mompati (RSM) District of North West Province, South Africa. RSM is comprised of both rural and peri-urban areas, with an economy centered on beef production and agriculture. The study area includes approximately 230,000 people, the majority of whom speak Setswana. North West Province has the fourth highest HIV prevalence in South Africa, with an estimated prevalence of 20.3% in the adult population 15–49 years  and 29.2The 2014 bio-behavioral survey employed multi-stage cluster sampling, with twenty-three enumeration areas (EAs) selected proportionate to size in each sub-district by Statistics South Africa (StatsSA) using 2011 census data. In each EA up to 36 inhabited dwelling units (DUs) were randomly selected from the StatsSA sampling frame for inclusion in the sample and one adult (18–49 years) was randomly selected per DU for participation. Local, trained fieldworkers assessed eligibility criteria, including age (18–49), residence in the home, and ability to consent; obtained written informed consent; and conducted a survey by computer-assisted personal interviewing in a private location at the participant’s home. Survey questions included HIV testing history, known status, history of HIV care and treatment, ART initiation, and medication adherence. The fieldworkers then referred consenting participants for HIV rapid testing and DBS sample collection. Full data collection procedures have been described elsewhere .For individuals consenting to HIV testing and sample collection, serostatus was determined using the Alere Determine HIV-1/2 rapid antibody test with finger-stick capillary blood  and, if reactive, confirmed using the First Response HIV 1–2.0 Rapid Whole Blood Test . Participants with HIV-positive or discrepant results were asked to provide finger-prick blood for DBS using a Munktell filter card . Participants who declined HIV rapid testing in their home could provide blood for DBS for laboratory HIV diagnosis (serology: ELISA confirmed with Western blot).Viral Load: DBS cards were dried, stored under desiccant at ambient temperature, transported to the testing laboratory  within six days of collection, and stored at − 70 °C. Viral load testing was performed using the COBAS AmpliPrep for sample preparation and COBAS TaqMan HIV-1 2.0 test  a previously validated method . We optehttp://hivdb.stanford.edu) [HIV Drug Resistance testing was performed using archived DBS with a VL > 5000 copies/mL at the National Institute for Communicable Diseases, Johannesburg, RSA. Spots were lysed in 2 ml of NucliSENS lysis buffer  for 2 h at room temperature. Total nucleic acid was extracted using the NucliSENS EasyMAG® automated system according to the manufacturer’s instructions. Amplification of a 1084 bp polymerase chain reaction (PCR) fragment consisting of codons 1–99 of protease (Pro) and codons 1–250 of reverse transcriptase (RT) was performed as previously described . Editingord.edu) .ART Drug Exposure was determined by a validated qualitative liquid chromatography-tandem mass spectrometry (LC MS/MS) method for the determination of the presence or absence of various antiretroviral analytes against cutoff samples analysed at a known concentration (0.02 μg/ml). A method was validated for the qualitative determination of 9 antiretroviral drugs from DBS, and consisted of a protein precipitation, followed by high performance liquid chromatography with MS/MS detection using a gradient elution. Deuterated internal standards were used for each analyte. An AB Sciex API 4000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of each of the protonated precursor ions. Analytes were assessed for the following three drugs: efavirenz (EFV), lopinavir (LPV), nevirapine (NVP), accounting for the majority of first and second line regimens, as tenofovir and emtricitabine are typically given in combination with efavirenz .Analyses aimed to characterize the scope of HIV drug resistance and types of DRM by treatment history among viremic HIV-positive participants. All analyses were weighted to account for sample design, with the exception of counts displayed in Fig. Virological failure was defined as ≥5000 copies/mL measured from DBS, a conservative cut-off because of the potential for contributions of pro-viral DNA in the quantification using DBS instead of plasma RNA , 34. We Overall 71.7% of participants consented to HIV-testing (749/1044). Of participants with confirmed HIV-positive status, 94% provided DBS (171/182), however one DBS card had insufficient specimen for genotyping. To assess whether the population with missing DBS data differed from the population with complete data, we used multiple imputation to create 50 copies of the dataset with simulated values based on observed data; our analyses with imputed datasets produced adjusted estimates for uncertainty. Missing HIV and viral suppression status were imputed based on age, sex, education, food insecurity, and lifetime number of sexual partners. Imputation of missing viral load also incorporated ART initiation, adherence, and months since diagnosis. All analyses were performed using Stata 14 .Among 182 seropositive participants confirmed by serial rapid testing or HIV DNA PCR, DBS were available for viral load and analyte testing from 170 participants , with greater sequencing failures among those who were positive for ART analytes compared to those who were ART-naïve or were negative for ART analytes but with reported prior ART exposure , abacavir (ABC), lamivudine or emtricitabine (XTC), tenofovir disoproxil fumarate (TDF), LPV/r and thus few would fail to suppress using the first-line regimens in South Africa. Among those who were analyte positive, over 10% and as high as 47% showed any resistance to XTC, TDF, EFV, NVP, and ABC and thus are predicted to show reduced susceptibility to first- or second-line regimes containing these antiretrovirals Fig. . Among tWe set out to examine the contribution of HIV drug resistance mutations to rates of virologic failure after noting that almost half of HIV-positive participants reporting ART initiation in a population-based sero-prevalence survey had evidence of virologic failure, despite 93% of those in care reporting near perfect adherence . In explLittle is known about the prevalence of DRM both prior to and following treatment initiation in the general population. One population-based study in South Africa found evidence of DRM in 7% of ART-naïve patients in rural KwaZulu-Natal in 2012, similar to our findings of DRMs in 8% of our 2014 North West population sequenced pre-treatment. Clinical cohort studies in sub-Saharan Africa have found a similar prevalence of drug resistance mutations in 6–11% of ART-naïve patients . If our Resistance was particularly high in our sample among those who had initiated ART, and most prevalent among those who had reported initiation but had no evidence of ART analytes, e.g. probable non-adherence. Non-adherence is likely a big contributor to lack of viral suppression and acquired resistance in this sample. It is not possible to distinguish between acquired and transmitted resistance in our data, however, patterns of resistance by ART status provide insights about contributions of acquired and transmitted resistance in this population-based sample. The large majority of DRMs in this population occurred in the ART-initiated population, implying a high likelihood of acquired resistance playing a central role, particularly with all mutations demonstrating NRTI/NNRTI (first-line) dual resistance among the analyte positive group. In fact, research has noted that a high proportion of adults and children who received first line ART will develop virological failure during the first 5 years, with 70–90% of patients in virologic failure acquiring drug-resistant HIV . While tThe extremely high rates of dual NRTI/NNRTI resistance among the analyte positive group has important implications for the future of the current first-line treatment. Currently the South African national program recommends that providers consider switching patients on the first-line drug regimen if the patient has experienced virological failure (VL > 1000 copies/mL) on at least two occasions two months apart despite good adherence. Our data indicate that approximately one-third of those who report ART intake are negative for analytes, making a premature decision to switch regimens potentially costly and could encourage further DRM. With South Africa currently starting up introduction of a new triple combination regimen of tenofovir disoproxil fumarate, lamivudine (3TC), and dolutegravir (TLD) , 38, somA strength of this study is the population-based sample in an understudied area with data on viral load, ART analytes, and DRMs. Our data also has several limitations. Only 70% of the samples were sequenced, with failure being highest for those not suppressed on ART and with comparatively lower viral load measures, which could lead to an underestimation of DRMs if that population is intermittently adherent. Studies restricted to ART-naïve populations using the same medium for genotyping have sequencing success of closer to 92% . AdditioWe found that both non-adherence and drug resistance mutations likely play a key role in virologic failure in this rural community in North West Province, South Africa. Increasing ART coverage can significantly lower the risk of new HIV infections in South Africa ; however


The integrase strand transfer inhibitor dolutegravir (DTG) has a high genetic barrier to resistance. Only rare cases of resistance to DTG have been reported when it is used as a component of antiretroviral therapy regimens in treatment-experienced patients unless there was prior use of a first-generation integrase inhibitor.A 38-year-old woman diagnosed with tuberculosis was switched to a second-line antiretroviral regimen of zidovudine, lamivudine and dolutegravir 50 mg 12-hourly together with rifampicin-based TB treatment. Based on treatment history and a previous resistance test there was resistance to lamivudine but full susceptibility to zidovudine. The patient did not suppress her viral load on this regimen and later admitted to only taking dolutegravir 50 mg in the morning because of insomnia.A second resistance test was performed which showed intermediate level of resistance to dolutegravir. Her regimen was changed to tenofovir, emtricitabine and ritonavir-boosted atazanavir with rifabutin replacing rifampicin for the remainder of her TB treatment. She achieved viral suppression on this regimen.To our knowledge this is the first case report from South Africa of emergent dolutegravir resistance in a treatment-experienced, integrase inhibitor-naïve patient. Factors that may have contributed to resistance emergence in this patient were that there was only one fully active nucleoside reverse transcriptase inhibitor in the regimen and lower exposure to dolutegravir because of the reduced dosing frequency while on rifampicin. A 38-year-old woman started antiretroviral therapy (ART) in 2007 with zidovudine (AZT), lamivudine (3TC) and efavirenz. Her baseline human immunodeficiency virus (HIV) viral load and cluster of differentiation 4 (CD4) cell count results were not available. In September 2009, she experienced virological failure , and a genotypic antiretroviral resistance test showed a thymidine analogue mutation , M184M/V and three non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations  see .Based on this genotype, her regimen was changed to tenofovir (TDF), emtricitabine (FTC) and ritonavir-boosted atazanavir. She then transitioned from HIV care in the private sector to the public sector where she was changed to abacavir (ABC), 3TC and ritonavir-boosted lopinavir. When she returned to private sector care in May 2017, she reported severe diarrhoea, resulting in poor adherence to ART. Her medication was switched to ABC, 3TC and ritonavir-boosted atazanavir and the diarrhoea settled.In June 2017, she was diagnosed with tuberculosis (TB) and was started on a rifampicin-based TB treatment. Her HIV viral load at this time was 345 406 copies/mL. As she already had significant diarrhoea on standard dose ritonavir-boosted lopinavir, it was felt that double-dose ritonavir-boosted lopinavir during TB treatment would not be tolerated. Given that AZT was still fully susceptible at the time of first-line failure, it was thought that AZT would still be susceptible and therefore providing one active nucleoside reverse transcriptase inhibitor (NRTI) to accompany dolutegravir (DTG). The patient was therefore switched to AZT, 3TC and DTG 50 mg 12-hourly. The patient informed her doctor in July 2017 that she was experiencing insomnia on this regimen. She was reassured and advised to continue until she had completed TB treatment.There were regular pharmacy claims for treatment from June 2017 until November 2017 when her viral load was 2800 copies/mL. A resistance test was performed in February 2018, and the following mutations were observed: three integrase mutations  resulting in high-level resistance to raltegravir and elvitegravir and intermediate resistance to DTG (based on the Stanford score), M184V and two NNRTI mutations (A98G and E138A). A sample was also collected for phenotypic testing at monogram and the results showed that there was a 21-fold reduction in DTG susceptibility. Furthermore, all the other available integrase inhibitors had reduced susceptibility .She then admitted to only taking DTG 50 mg in the morning because she had experienced insomnia with twice daily dosing, which resolved if she skipped the evening dose. Based on the resistance test result, her ART regimen was changed to TDF, FTC and ritonavir-boosted atazanavir in February 2018 with rifabutin replacing rifampicin for the remaining months of TB treatment. An HIV viral load performed 3 months after the change was 230 copies/mL, and subsequent viral loads have been below 20 copies/mL for about 12 months.1The integrase strand transfer inhibitor DTG has a high genetic barrier to resistance. In clinical trials evaluating DTG as a component of triple drug therapy in ART-naïve patients, no emergence of resistance to DTG has been reported. Only rare cases of emergence of resistance to DTG have been reported when it has been used as a component of ART regimens in treatment-experienced patients, unless there has been prior use of the first-generation integrase inhibitors, raltegravir and elvitegravir.In the case reported here of a treatment-experienced patient, DTG resistance was detected after 8 months on a regimen of AZT, 3TC and DTG. Based on the patient’s treatment history and resistance test results, this patient’s virus (at the time of starting this regimen) had resistance to 3TC , but was fully susceptible to AZT. The Stanford score for AZT on the 2009 resistance test was 0  and between 2009 and 2017, the patient received only TDF/FTC and ABC/3TC as NRTIs. These NRTI combinations typically do not select for TAMs, the mutations that may compromise AZT. In addition, there were no TAMs found on the 2018 resistance test.2 Whilst no protease inhibitor mutations were detected in patients in the ritonavir-boosted lopinavir arm, 2/283 patients in the per-protocol analysis and in the DTG arm developed integrase resistance mutations . One patient developed H51HY, G118R, E138EK and R263K mutations and the other patient developed the G118R mutation. Like these two cases in DAWNING, the patient reported here developed DTG resistance on second line despite there being a fully active NRTI in the regimen.Based on the Dolutegravir versus ritonavir-boosted lopinavir both with dual nucleoside reverse transcriptase inhibitor therapy in adults with HIV-1 infection in whom first-line therapy has failed (DAWNING) trial results, it would be predicted that her regimen of DTG with two NRTIs, one of which (AZT) was fully active, should be effective. The DAWNING trial evaluated DTG with NRTIs in second-line ART and demonstrated that when there was at least one fully active NRTI accompanying DTG, then this regimen was superior to ritonavir-boosted lopinavir combined with NRTIs: at week 48, 84% in the DTG arm achieved viral suppression compared with 70% in the lopinavir arm.3 Studies in healthy volunteers and patients with HIV and TB have shown that this reduction can be compensated by increasing the frequency of DTG dosing from 50 mg daily to 50 mg 12-hourly in patients on rifampicin, which results in trough concentrations similar to patients taking DTG 50 mg daily without rifampicin, and resulted in adequate virological responses in patients with HIV and TB in the INSPIRING trial.5 Our patient decreased the DTG dose to 50 mg daily whilst on rifampicin because of insomnia, which would have resulted in considerably lower exposure to DTG and may have contributed to the emergence of resistance. A case has been reported in which DTG resistance emerged in a patient on DTG-containing first-line regimen who was taking rifampicin for treatment of a staphylococcal infection. The DTG was increased to 50 mg twice daily, but despite this, the patient had unexpectedly lower DTG concentrations.6Another factor that may have contributed to the development of DTG resistance in this patient was the drug interaction with rifampicin. Rifampicin is a potent inducer of UGT1A1 and CYP3A4, the enzymes that metabolise DTG and thereby reduces concentrations of DTG (the trough DTG concentration is reduced by 85%).8 it is possible that a dose increase is not required in patients on first-line DTG-based ART and rifampicin. This is supported by a recent pharmacokinetic study in healthy volunteers that showed that in patients taking DTG 50 mg once daily with rifampicin, all had DTG trough concentrations above the protein-adjusted IC90.3 A recent observational study from Botswana reported similar virologic outcomes in patients on rifampicin taking DTG 50 mg 12-hourly versus 50 mg daily.9 The RADIANT-TB trial is being conducted in Cape Town and is enrolling patients on TB treatment and starting first-line ART who are being randomised to TDF/FTC/DTG 50 mg 12-hourly versus TDF/FTC/DTG 50 mg daily . Until data from this trial are available, we recommend DTG be dosed at 50 mg 12-hourly in all patients on rifampicin.It is important to note that the need for this dose adjustment of DTG with rifampicin is currently being investigated. Because DTG was shown to have antiviral efficacy at doses as low as 10 mg daily in phase 2 trials,11 This may also have been a contributing factor in the patient described in this case report.Whilst very few cases of DTG resistance have been reported in patients taking DTG as part of a triple-drug first-line ART regimen in routine clinical practice, one feature of the cases reported has been a high baseline viral load.12 there is limited data on the risk of DTG resistance in treatment-experienced, integrase inhibitor-naïve patients, especially in routine clinical practice, with concomitant usage of rifampicin and variable adherence and when used long term. To our knowledge, this is the first case report from South Africa of DTG resistance emerging in a patient who was integrase inhibitor-naïve when starting DTG. Factors that may have contributed to resistance emergence in this patient were that there was only one fully active NRTI in the regimen in a treatment-experienced patient (based on population-based genotyping) and the lowered exposure to DTG because the patient reduced the DTG dosing frequency from 50 mg 12-hourly to 50 mg daily against her doctor’s advice whilst on rifampicin because of insomnia.In conclusion, although there is a very low risk of DTG resistance when used as part of the first-line combination therapy, DTG resistance is not uncommon after monotherapy and when used in patients previously exposed to raltegravir or elvitegravir therapy. However, apart from the DAWNING and Dolutegravir versus raltegravir in antiretroviral-experienced, integrase-inhibitor-naive adults with HIV (SAILING) trials,


Half of the patients  harboured genotypic drug resistance, with M184V  and K103N  being the most prevalent mutations. No significant difference in the median time to re-suppression (31–39 weeks) were observed for either group (p = 0.41). However, re-suppressors with mutant virus rebounded significantly earlier than those with wild-type virus . Similar phenotypic drug resistance profiles were observed between patients who re-suppressed and patients who failed to re-suppress. While most remained susceptible to stavudine (d4T) and zidovudine (AZT), both groups showed a reduced susceptibility to 3TC and NNRTIs. HIV- 1 infected patients on an NNRTI-based regimen can achieve viral re-suppression on the same regimen despite harbouring viruses with genotypic and phenotypic drug resistance. However, re-suppression was less durable in those with resistance, reinforcing the importance of appropriate regimen choices, ongoing viral load monitoring and adherence counselling.We have previously reported on HIV-1 infected patients who fail anti-retroviral therapy but manage to re-suppress without a regimen change despite harbouring major drug resistance mutations. Here we explore phenotypic drug resistance in such patients in order to better understand this phenomenon. Patients (n = 71) failing a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen, but who subsequently re-suppressed on the same regimen, were assessed for HIV-1 genotypic drug resistance through Sanger sequencing. A subset (n = 23) of these samples, as well as genotypically matched samples from patients who did not re-suppress (n = 19), were further assessed for phenotypic drug resistance in an  The use of antiretroviral therapy (ART) has a significant impact on the control of HIV-1 infection and HIV associated morbidity . South AThe South African national guidelines for the management of HIV infection promote viral load testing for monitoring viral suppression on ART, as well as for diagnosing treatment failure . At the in vitro phenotypic resistance testing to evaluate genotypic drug resistance in the context of re-suppression and failure.Re-suppression has been observed in patients with major genotypic drug resistance mutations particularly those failing an NNRTI-based first-line regimen with M184V and K103N , 13, 15.At the time of initiation of ART, patients were offered the opportunity to participate in the evaluation cohort. Patients with CD4 count <250 cells/mm3; WHO stage 3 and CD4 count <350 cells/ mm3; or WHO stage 4, who initiated ART, were included in the overall cohort. CD4 counts and HIV RNA levels (VL) were determined before initiation, after 6 weeks on ART and every 6 months thereafter. Patients with a detectable viral load of >1,000 copies/ml after previously being undetectable, a sustained increase in viral load of >0.6 log from its lowest point and a return to 50% of the pre-treatment viral load were eligible to switch to a PI-based second line regimen. Due to concerns regarding adherence, tolerability of the second-line regimen, and premature regimen switching many patients where not switched to a second-line regimen until multiple elevated viral loads and, often, a notable CD4 count decline ) or AZT/3TC (15/71 [21%]) containing regimen receiving either EFV (41/68) or NVP (27/68) The remaining 4% (3/71) of patients were on a regimen of TDF/FTC/EFV, TDF/FTC/NVP or ABC/3TC/EFV, respectively.Almost all patients (70/71 [99%]) were infected with HIV-1 subtype C. One patient was infected with an HIV-1 CRF02_AG recombinant strain. Genotypic resistance analysis indicated that half of the patients (36/71 [51%]) contained virus with major genotypic drug resistance mutations : NNRTI m10 copies/ml; IQR 1.39–3.32) during the full monitoring period compared to patients with wild-type virus . However, at the sampling time point, viral loads were significantly (p = 0.017) lower among those with mutant viruses (3.89 log10 copies/ml vs. 4.35 log10 copies/ml). Treatment responses after the sampling point showed that patients with mutant viruses had lower rates of virological re-suppression (to VL<400: 23 [64%]), higher rates of transient viremia  and more repeat failures  than those with wild-type virus .Patients with mutant virus had a significantly (p = 0.005) higher median VL (2.31 logPatients with mutant viruses were monitored for significantly (p = 0.020) longer periods  than those with wild-type virus . No significant difference (p = 0.508) was observed in the median time between visits for those with wild-type  and mutant virus . Sampling time points were significantly (p = 0.047) later for those with mutant virus  than for those with wild-type virus . No significant difference (p = 0.408) was observed in the median duration of re-suppression until repeat failure between those with wild-type  and mutant  virus. The maximum time of re-suppression was 116 and 98 weeks for wild-type and mutant samples, respectively (not shown). For patients with a subsequent repeat failure, a significant difference (p = 0.014) was observed in time to the repeat failure for those with wild-type  and mutant  virus.From the 36 patients who re-supressed on the same regimen in the presence of one or more major NRTI and/or NNRTI mutations, 21 patients with various degrees of genotypic drug resistance  representative of the cohort were selected for further phenotypic analysis. As a comparator group, 18 patients with similar drug resistance mutation profiles, who did not re-suppress or who had an in-class drug switch before re-suppression, were included (termed failing patients). For both groups, genotypic resistance patterns varied with some patients having single mutations and others with more complex combinations of up to 9 mutations . Two re-10 copies/ml; IQR 2.98–3.98) during the entire monitoring period than those who re-suppressed . Higher median viral loads were significantly associated with failure . There was no significant difference (p = 0.944) in the median viral loads at the sampling points. A significantly (p<0.0001) higher number of patients who failed  had breakthrough events before the sampling point, compared to patients who re-suppressed . We found that the number of previous breakthrough events were significantly predictive of failure . Both groups of patients were on similar regimens and displayed similar genotypic resistance profiles, with the K103N (≥57%) and M184V (≥71%) mutations being the most prevalent.When comparing the re-suppressor subgroup (n = 21) to the failing patients (n = 18), both groups had a median time between HIV RNA assays of ~24 weeks (IQR 20–27 weeks) and were followed over a similar time period of 193–243 weeks . Howeverin vitro phenotypic drug susceptibility against the ARVs that matched the regimen of the corresponding patients  while remaining fully or partially susceptible to only one antiretroviral drug (d4T or AZT). We found that the phenotypic response to ARVs was not predictable of failure, but that the number of previous viral breakthroughs were. These findings suggest that adherence, rather than viral susceptibility to a sub-optimal ART regimen, may be the determining factor with regards to re-suppression and failure.We found that half of the patients that re-suppressed after virologic breakthrough contained major NRTI (e.g. M184V) and NNRTI  drug resistance mutations, typically observed in patients failing an NNRTI-based regimen , 23. TheIt is well described that non-adherence is linked to the development of drug resistance and HIV disease progression . FurtherThe detection of early virological failure should provide an opportunity to increase adherence counselling and repeat viral load testing before a switch in treatment . EnhanceNon-adherence is multifactorial and solutions to circumvent this are complex . Effortsin vitro phenotypic assay, an assumption was made that FC values above the TCO values translated into a decrease in drug susceptibility. This could potentially have lead to the under- or over-estimation of phenotypic drug resistance in the patient samples. Lastly, although we used drug level testing as a surrugate for adherence, no reliable adherence data was available for use in this study and could potentially limit the conclusions that were drawn. In addition, CD4+ T-cell counts and pre-treatment viral loads are important factors that could contribute to viral failure 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: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********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: The study titled “HIV-1 re-suppression on a first-line regimen despite the presence of phenotypic drug resistance” investigates the phenotypic drug resistance in HIV-1 infected patients who fail anti-retroviral therapy but re-suppress on the same regimen in spite of possessing drug resistance mutations. The study and the experiments were designed well, although the authors did not find any significant differences in the phenotype resistance patterns between the study group and the comparator group. The authors, however, report that the number of previous viral breakthroughs, and therefore, adherence to treatment regimen, may be the determining factor for re-suppression and failure. The manuscript was well-written.Minor Comment1. In lines 239 and 240, the authors have selected 21 patients for phenotypic analysis out of the 36 patients who were re-suppressed on the same regimen in the presence one of more major NRTI and/or NNRTI mutations. The authors have not explained why all the 36 patients were not considered for the phenotypic analysis or what criteria was used to select the 21 patients. Please elaborate.**********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,  13 May 2020Reviewer 1: Minor CommentIn lines 239 and 240, the authors have selected 21 patients for phenotypic analysis out of the 36 patients who were re-suppressed on the same regimen in the presence one of more major NRTI and/or NNRTI mutations. The authors have not explained why all the 36 patients were not considered for the phenotypic analysis or what criteria was used to select the 21 patients. Please elaborate.Response: We have now clarified this in the text:Page 12, lines 262 – 266: “From the 36 patients who re-supressed on the same regimen in the presence of one or more major NRTI and/or NNRTI mutations, 21 patients with various degrees of genotypic drug resistance  representative of the cohort were selected for further phenotypic analysis.”AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 5 Jun 2020HIV-1 re-suppression on a first-line regimen despite the presence of phenotypic drug resistancePONE-D-20-09445R1Dear Dr. Basson,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Cristian Apetrei, MD, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 9 Jun 2020PONE-D-20-09445R1 HIV-1 re-suppression on a first-line regimen despite the presence of phenotypic drug resistance Dear Dr. Basson:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Cristian Apetrei Academic EditorPLOS ONE


The anti-retroviral combination of abacavir/lamivudine plus rilpivirine (ABC/3TC/RPV) is not recommended by international guidelines as the first-line regimen. However, it is potent, well-tolerated, and affordable, especially in resource-limited settings. This study evaluates the efficacy and safety of ABC/3TC/RPV as an initial regimen for treatment-naïve HIV-1 infected patients.A retrospective study was conducted in the largest HIV care centre in Singapore, with data collected June 2011 to September 2017. All treatment-naïve HIV-1 infected adults prescribed ABC/3TC as part of their initial anti-retroviral therapy regimen were included. The third drug was a non-nucleoside reverse-transcriptase inhibitor (NNRTI) such as RPV or efavirenz (EFV), or boosted protease-inhibitor (PI). Patients were followed up for 48 weeks. The primary end-point was the percentage of patients achieving virologic suppression, analysed using on-treatment analysis. Secondary outcomes included CD4-count change, treatment discontinuation and treatment-related adverse events.170 patients were included in the study, 66 patients in the RPV group, 104 patients in the comparator group (EFV or boosted PI). 96% (n = 24) in the RPV group and 87% (n = 26) in the comparator group achieved viral suppression at 48 weeks (p = 0.28). Median (interquartile range) time to viral suppression was similar: 17 (14–24) weeks in the RPV group, and 21 (13–26) weeks in the comparator group. There were no statistically significant differences in the CD4 count between the two groups. 14% (n = 9) of patients on RPV discontinued treatment before 48 weeks, compared to 30% (n = 31) from the comparator group (p = 0.053). Of these, 23 discontinuations were due to drug adverse effects, and only 1 attributed to RPV (p < 0.01). One patient in each group had virologic failure.RPV is effective, safe and considerably more tolerable than compared to NNRTI or boosted PI in ABC/3TC-containing regimens for treatment-naïve patients. It offers an affordable and attractive option, especially in resource-limited settings. Current combination anti-retroviral therapies (cART) against human immunodeficiency virus (HIV) are highly effective. Conventional three-drug cART regimens for treatment naïve patients generally consist of two nucleoside reverse transcriptase inhibitors (NRTIs) and a third drug from another class: integrase strand transfer inhibitors (INSTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) or protease inhibitors (PI). In 2019, the World Health Organisation (WHO) updated its recommendations on the preferred first-line HIV regimen-favouring INSTIs in combination with two NRTIs . These cNRTI backbone therapies include tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) (a prodrug formation of TDF) in combination with emtricitabine (FTC) or lamivudine (3TC), and abacavir and lamivudine (ABC/3TC). ABC/3TC offers the main advantage of avoiding potential renal and bone toxicity seen with tenofovir based regimen and can be used in individuals with renal insufficiency and osteoporosis , 6. It i3 [Rilpirivine (RPV) is a second-generation NNRTI approved for use with NRTIs in treatment-naïve HIV-1 patients with pre-treatment viral loads of less than 100,000 copies/ml , 3. Its 3 , 3. RPV 3 , and a m3 . The com3 . This coHowever, data on the use of ABC/3TC in combination with RPV in treatment-naïve patients are scarce. Most patients in the ECHO and THRIVE clinical trials received TDF/FTC as the NRTI backbone, and only 35 patients in the THRIVE study received ABC/3TC plus RPV –11. CurrThe purpose of this study is to evaluate the efficacy and safety of ABC/3TC plus RPV as an initial regimen for treatment-naïve HIV-1 patients in routine clinical practice in Singapore.3, hepatitis B co-infection (requiring treatment with TDF) and pregnancy.This was a retrospective study conducted at the National Centre for Infectious Diseases (NCID), which provides HIV care for the largest cohort of HIV-infected individuals in Singapore, between June 2011 and September 2017. All treatment-naïve HIV-1 infected patients (≥ 18 years old) who received ABC/3TC were included. The third drug could either be a NNRTI (RPV or EFV) or boosted PI (atazanavir or darunavir). Exclusion criteria were pre-treatment viral load > 100,000 copies/ml, CD4 count < 200 cells/mmData were collected from the NCID HIV Clinical Database, a standardised computerised database containing records of demographic information, HIV transmission route, baseline viral load and CD4 count, AIDS-defining illnesses, cART regimen history and treatment-associated adverse events. Individual patient case records were reviewed to determine underlying medical conditions and to obtain laboratory results such as lipid profile, kidney and liver function tests when these were not available on the Clinical Database. Patients were followed up for 48 weeks from initiation of cART. The interval for follow-up was determined by individual attending physicians as per routine clinical care guidelines. During follow-up, information regarding viral load, CD4 count, laboratory parameters, treatment-related adverse events, and reasons for discontinuing cART, if any, were collected. If cART was stopped due to virologic failure, genotype resistance testing (GRT) was done. Data were anonymised upon extraction. The follow-up was censored for analyses in March 2018.Virologic response was classified according to the definitions in the DHHS guidelines . VirologThe primary efficacy end-point was the percentage of patients who achieved virologic suppression . This outcome was analysed using on-treatment analysis, in which discontinuation and missing data were censored. The median time taken to achieve virologic suppression was also analysed. Secondary outcomes were changes in CD4 count, treatment discontinuation before 48 weeks, reasons for discontinuation, treatment-related adverse events, and lipid, kidney and liver profile changes.Univariate analyses were carried out using the Chi square and Fisher’s exact test for categorical variables. Kaplan–Meier survival curves were drawn to show median time to HIV viral load suppression, for the 2 treatment regimes. Log-rank tests were used to test for differences between the 2 survival functions. Cox proportional hazards model was used to obtain the treatment effect sizes. When necessary, follow-up was categorised into 12-weekly intervals. Using Stata MP 13 , all analyses were performed at a 5% significance level.3 (n = 19). Sixty-six patients (39%) received ABC/3TC plus RPV and 104 patients (61%) received ABC/3TC plus EFV or a boosted PI , pre-treatment viral load > 100,000 copies/ml (n = 19) and CD4 count < 200 cells/mmThere were no significant differences in the baseline characteristics between the RPV group and the EFV or boosted PI group Table . Median 3 to 583 (513–660) cells/mm3 and in the EFV or boosted PI group, from 394 (297–511) cells/mm3 to 574 (479–665) cells/mm3 over the 48-week period  in the RPV group and 87% (n = 26) in the EFV or boosted PI group, p = 0.28  from the RPV group had discontinuation of treatment before 48 weeks, compared with 31 out of 104 patients (30%) from the EFV or boosted PI group (p = 0.05). In the RPV group, 1 patient discontinued treatment due to an adverse reaction, 1 had virologic failure, 3 defaulted clinic follow-up and 4 had other reasons for discontinuation. In the EFV or boosted PI group, the main reason for discontinuation was adverse reactions . One patient had virologic failure, 4 defaulted clinic follow-up, 1 had cART regimen switched based on a decision made by the attending physician, 1 patient requested for cART switch, 1 patient died and 1 had other reasons for discontinuation (Fig. 4% from tA total of 23 patients discontinued their initial cART due to adverse reactions (Table Two patients fulfilled the DHHS guideline criteria for virologic failure. One patient who received ABC/3TC plus RPV had a viral load of 9730 copies/ml at 24 weeks and 26,200 copies/ml at 32 weeks. GRT showed multiple NRTI, NNRTI and PI associated mutations. These included M41L, D67N, M184V, T215Y, K101KE, E138EAG, Y181C, Y188YFHL, and M230ML mutations, many which are associated with high levels of reduced susceptibility to ABC/3TC as well as RPV [Three patients had virologic rebound: 2 from the EFV or boosted PI group and 1 from the RPV group. The first patient had a viral load of 35,136 copies/ml at 48 weeks after achieving virologic suppression previously. He was non-adherent to EFV because of giddiness and drowsiness, and EFV was switched to ritonavir-boosted lopinavir, achieving undetectable viral load thereafter. His GRT showed K103N, reflecting a reduced 20-fold reduced susceptibility to EFV [There were no significant changes in the lipid, kidney and liver profiles at 48 weeks between the two groups Fig. .Fig. 5Ch3. We report treatment efficacy of 96% at 12 months, which is similar to previous studies in the published literature. In addition, there was no significant difference in the virologic effectiveness compared with the EFV or boosted PI group.Data on ABC/3TC plus RPV as initial regimen in treatment-naïve HIV-1 infected patients are scarce. In the THRIVE study, only 35 patients received ABC/3TC plus RPV, with an overall efficacy of 86%, without apparent differences between different NRTI backbones . In 2016Our study also showed that ABC/3TC plus RPV is a safe and tolerable regimen with fewer adverse reactions and subsequent treatment discontinuations compared with ABC/3TC plus EFV or boosted PI. Most discontinuations were seen in patients receiving ABC/3TC plus EFV. The main reason was neuropsychiatric side effects from EFV such as abnormal dreams, insomnia, headache, giddiness and depression, which are well-described and have prompted the EFV-based cART regimens to be recommended as alternative regimens by the DHHS and other guidelines , 14. RPVA significant advantage of using ABC/3TC plus RPV is its once-daily dosing frequency, proffering a regimen with low pill burden and small pill size. It consists of two tablets taken daily, with RPV being one of the smallest ART tablet available at 6.4 mm in diameter . MoreoveImportant considerations when using RPV include that it needs to be taken with meals to ensure adequate bioavailability and to avoid co-administration with drugs that reduce gastric acidity such as proton pump inhibitors as RPV absorption is dependent on gastric pH , 16. TheOur study has several limitations. It was a retrospective, single centre study with relatively small sample size, and there may have been systematic differences in the two groups that could have served as confounders for the observed differences in outcome. We note a predominance of males in our study population, which reflects the general HIV epidemiology in Singapore, however it may not be representative of other populations. Furthermore, not all patients had the same duration of follow-up or interval between follow-up visits. This reflects variation in clinical practice among clinicians. Despite this, the median time to virologic suppression was approximately 17 weeks for the RPV group and 21 weeks for the EFV or boosted PI group. Longer follow-up of these patients would allow better evaluation of the long-term effectiveness and safety of this regimen. It is also imperative to note the limitations of this regimen: reduced efficacy in patients with high pre-treatment viral load and low CD4 count; the lack of safety data in pregnancy; and potential interactions with commonly used medications such as rifampicin and proton-pump inhibitors.In conclusion, we present the first analysis that directly compared ABC/3TC plus RPV versus ABC/3TC plus EFV or boosted PI as a first-line regimen in treatment-naïve HIV-1 patients. We found similar efficacy and safety of RPV in combination with ABC/3TC in the clinical setting (Additional file Additional file 1: Table S1. Baseline characteristics of patients in the RPV, EFV and boosted PI groups.


IP-10/CxCL-10 was significantly lower at follow-up in responders versus poor-responders . KSHV nAb were not significantly differential between responders and poor-responders. In conclusion, high plasma IL-5 at baseline could be a marker for ART-treated KS tumor regression, whereas increased pro-inflammatory cytokine IL-6, and the chemokine IP-10, associate with KS tumor progression.HIV-associated/epidemic Kaposi’s sarcoma (EpKS) is an AIDS-defining angio-proliferative malignancy. It can be treated with antiretroviral therapy (ART) alone or with ART plus cytotoxic chemotherapy. ART-treated EpKS can either respond or worsen upon treatment. This study aimed at identifying immunological markers of ART-treatment response. We compared responders  versus poor responders (those with progressive or non-responsive EpKS). We measured plasma cytokine and chemokine levels using cytometric bead assays. Kaposi’s sarcoma herpesvirus (KSHV) neutralizing antibody (nAb) responses were also quantified to test associations with treatment outcome. Interleukin (IL)-5 levels were significantly elevated in responders versus poor-responders at baseline (0.76pg/ml  Kaposi’s sarcoma (KS) is a vascular malignancy highly prevalent in sub-Saharan Africa (SSA) –3. HumanIt is clear from the epidemiological association of KS with HIV-induced immunosuppression, immunosuppressive therapy, and aging –8 that iManagement of EpKS can be challenging due to difficulties in disease staging, adverse effects of available treatments, and poor outcomes. Early-diagnosed skin-limited EpKS is usually treated with ART alone, whereas advanced EpKS requires ART plus cytotoxic chemotherapy. Treatment of EpKS with ART results in variable outcomes including remission, stable disease, or progression requiring addition of cytotoxic cancer chemotherapy. Furthermore, EpKS patients initiating ART have increased risk of mortality when compared with non-KS HIV-infected individuals initiating ART . CytotoxCytokines have been observed to be dysregulated in KS patients –15. AntiThis study was conducted on adult KS patients presenting at the University Teaching Hospital (UTH) in Lusaka, Zambia. We recruited histologically-confirmed, early-stage  HIV-associated KS patients who were antiretroviral therapy (ART)-naïve or on ART for less than 2 weeks. Recruitment was done after obtaining informed consent. All recruited patients were 18 years and older, had a limited number of early KS lesions , lacked lymphedema, and had no evident visceral involvement. At baseline, sociodemographic information was collected, a physical assessment was conducted, and blood samples were collected. ART was initiated in those who were ART-naïve. All recruited patients were followed up monthly for at least 6 months for either remission (responders) or progression of KS (poor responders). Responders all had a follow up period of at least 6 months while poor responders had variable follow up ranging from 1 to 6 months depending on when they experienced disease progression. None of our study participants had co-infections such as malaria or hepatitis, or HIV-associated comorbidities such as tuberculosis or cryptococcal meningitis at baseline or during follow up. Venous whole blood was collected at baseline and at the time of progression or after 6 months for those who responded to ART. All blood samples were collected in EDTA vacutainers. Plasma was separated and then stored at -80°C until analysis. The study was approved by the University of Zambia Biomedical Research Ethics Committee, the Zambian National Health Research Authority, and the Institutional Review Board at the University of Nebraska-Lincoln.HIV-1 status for all patients was determined as recommended by the Zambian Ministry of Health. The Alere Determine HIV-1/2 kit  was used for screening, while the SD Bioline HIV-1/2 kit (Standard Diagnostics Inc) was used for confirmation. HIV-1 plasma viral load was measured on the Hologic Panther (Hologic) using the Aptima HIV-1 Quant Dx Assay kit (Hologic) according to the manufacturer’s protocol.CD4 counts were determined with BD FACSCalibur (BD Biosciences) using the BD TriTest kit (BD Biosciences), according to the manufacturer’s protocol.Plasma cytokines and chemokines were quantified using the Beckton-Dickinson Cytometric Bead Array (CBA) Flex Set kits according to the manufacturer’s protocol. The following cytokines and chemokines were quantified: Transforming growth factor-β (TGF-β), Interleukin 5 (IL-5), Interferon-inducible protein 10 (IP-10), Vascular endothelial growth factor (VEGF), Interleukin 6 (IL-6), Tumor Necrosis Factor (TNF), and Interleukin 10 (IL-10). Data was quantified on a BD Accuri C6 Plus cytometer at 448nM and 640nM excitation wave lengths  and analyzed with FlowJo version 10 software .The challenge virus, KSHV (rKSHV.219) encoding the green fluorescent protein (GFP) gene under control of the cellular EF1α promoter, was generated by stimulating latently infected Vero.219 cells, as previously described , 16, 17.Baseline characteristics were analyzed using descriptive statistics. The Kruskal-Wallis test was used to compare cytokine/chemokine differences between groups. Comparison of paired data within groups was done using the Wilcoxon matched-pairs signed-rank test. Testing for correlation of continuous variables was done using the Spearman rank correlation. All statistical tests were two-sided, p values <0.05 were considered significant. Stata version 15  was used for all statistical analyses.We successfully recruited 27 eligible study participants. 5 of the recruited participants were lost to follow up after the baseline assessment, 1 participant was commenced on cytotoxic chemotherapy at another health facility and hence was no longer eligible for follow up, 13 participants had worsening KS within 6 months of ART initiation, and 4 participants had stable disease or regression of KS lesions after at least 6 months of follow up. Among the participants that worsened on ART, only those that were on ART for at least 2 months before worsening of KS were analyzed. We therefore analyzed 11 EpKS patients who had early-stage KS disease and recently initiated ART, 7 of these had EpKS disease progression within 6 months of initiation of ART and follow up while 4 had stable disease or KS tumor regression for at least 6 months. Among the poor responders, the median HIV plasma viral load at baseline was 45,610 copies/ml  while at determination of ART response (DAR) the median HIV viral load had dropped to 82 copies/ml [IQR = 0–2664]. Responders had a baseline median HIV viral load of 577,016 copies/ml  which dropped to 353 copies/ml [IQR = 15–1679] at DAR. There was no statistically significant difference in HIV viral load at baseline or at DAR between the responders and poor responders. There was a statistically significant decrease in HIV viral load from baseline levels at DAR among the poor responders (p = 0.018), whereas the drop in HIV viral load among the responders was not statistically significant (p = 0.11).The median CD4 count for the poor responders was 166 cells/μl [IQR = 127–295] at baseline, and increased to 195 cells/μl [IQR = 131–339] at DAR. The median CD4 count for responders was 135 cells/μl [IQR = 68–211] at baseline and was 135 cells/μl [IQR = 101–206] at DAR. There was no statistically significant difference in CD4 counts at baseline and at DAR between responders and poor responders. In addition, CD4 count comparisons between baseline and DAR in both responders and poor responders were not statistically significant.We quantified the plasma levels of TGF-β, IL-5, IP-10, VEGF, IL-6, TNF, and IL-10 in responders and poor-responders at baseline and at DAR. These cytokines and chemokines have previously been associated with regulation of cell growth and survival, humoral immune response, chemotaxis, tumor proliferation, and enhancing and/or suppressing cancer-associated inflammation –22. TherThe median plasma level of the chemokine CXCL10 (IP-10) was not significantly differential at baseline between responders and poor responders  . HoweverKS patients have much higher neutralizing antibody (nAb) responses than KSHV-infected, but asymptomatic individuals with HIV-1 co-infection , 16. HowHIV-induced immune suppression and dysregulation are major predisposing factors for the development of EpKS. However, it is not entirely clear what HIV does and to what extent. Therefore, treatment with ART is essential in the management and long-term control of KS. ART-induced immune reconstitution reverses KS progression . HoweverWe observed a decrease in HIV viral loads in both responders and poor responders to viral suppression levels of less than 1000copies/ml. Previous studies have reported that ART has no direct anti-KSHV or anti-KS activity . In addiConsistent with previous reports of KSHV nAb primarily in KS symptomatic subjects , 16, botWe did, however, observe differences in IL-5, IL-6 and IP-10 between responders and poor responders. IL-5 is a Th2 cytokine that induces eosinophilia and also promotes immunoglobulin secretion by B cells . IL-5 leWe also observed higher plasma IL-6 levels in individuals who were undergoing KS disease progression compared to those who underwent response. IL-6 is a pleiotropic cytokine known to stimulate the proliferation of KSHV-infected cells , 32, andIP-10 is a chemoattractant for recruiting leukocytes to involved tissues, and thereby intensifies inflammation that results in tissue damage , 40. It The major limitation of this study was the low number of complete responders. A larger sample size with increased numbers of both responders and poor responders, and a longer follow up period would be required in future.High plasma IL-5 is a potential marker of good prognosis in ART-treated EpKS, whereas high plasma IL-6 and IP-10 levels are prognostic markers for potentially poor ART-treatment outcomes.