Incomplete adherence to antiretroviral therapy (ART) results in virologic failure and resistance. It remains unclear which adherence measure best predicts these outcomes. We compared six patient-reported and objective adherence measures in one ART-naïve cohort in South Africa.We recruited 230 participants from a community ART clinic and prospectively collected demographic data, CD4 count and HIV-RNA at weeks 0, 16 and 48. We quantified adherence using 3-day self-report (SR), clinic-based pill count (CPC), average adherence by pharmacy refill (PR-average), calculation of medication-free days (PR-gaps), efavirenz therapeutic drug monitoring (TDM) and an electronic adherence monitoring device (EAMD). Associations between adherence measures and virologic and genotypic outcomes were modelled using logistic regression, with the area under the curve (AUC) from the receiver operator characteristic (ROC) analyses derived to assess performance of adherence measures in predicting outcomes.At week 48 median (IQR) adherence was: SR 100% (100–100), CPC 100% (95–107), PR-average 103% (95–105), PR-gaps 100% (95–100) and EAMD 86% (59–94), and efavirenz concentrations were therapeutic (>1 mg/L) in 92%. EAMD, PR-average, PR-gaps and CPC best predicted virological outcome at week 48 with AUC ROC of 0.73 (95% CI 0.61–0.83), 0.73 (95% CI 0.61–0.85), 0.72 (95% CI 0.59–0.84) and 0.64 (95% CI 0.52–0.76) respectively. EAMD, PR-gaps and PR-average were highly predictive of detection of resistance mutations at week 48, with AUC ROC of 0.92 (95% CI 0.87–0.97), 0.86 (0.67–1.0) and 0.83 (95% CI 0.65–1.0) respectively. SR and TDM were poorly predictive of outcomes at week 48.EAMD and both PR measures predicted resistance and virological failure similarly. Pharmacy refill data is a pragmatic adherence measure in resource-limited settings where electronic monitoring is unavailable.Trial registration The trial was retrospectively registered in the Pan African Clinical Trials Registry, number PACTR201311000641402, on the 13 Sep 2013 (www.pactr.org). The first participant was enrolled on the 12th July 2012. The last patient last visit (week 48) was 15 April 2014The online version of this article (doi:10.1186/s12981-017-0138-y) contains supplementary material, which is available to authorized users. Adherence is critical to realising the clinical and prevention benefits of ART –3. DespiA variety of ART adherence measures have been used in both observational and intervention studies. Self-report is the most frequently used method, but is often imprecise and overestimates adherence, similar to clinic-based pill count –7. PharmFew studies have directly compared the ability of multiple adherence monitoring methods to predict virological failure in one cohort –17 or exOur study compared a variety of adherence measures, three widely accessible to clinicians in a resource-poor setting: self-report (SR), clinic-based pill count (CPC) and pharmacy refill data (PR-average); and three requiring additional technology or calculation: therapeutic drug monitoring (TDM), calculation of medication free days or gaps (PR-gaps) and real-time electronic drug monitoring. All adherence measures were collected in a prospective ART-naïve cohort starting first-line ART, in order to explore which measure best predicted virological or resistance outcome at 16 and 48 weeks into treatment.This study is a sub-study of a randomised controlled trial recruited at the Hannan Crusaid Treatment Centre, an outpatient antiretroviral treatment centre in Cape Town, South Africa. This site and the study have been described elsewhere , 20. TheCounts of pill returns were done at each scheduled clinic visit. Those with a raised HIV-RNA (>1000 copies/ml) or non-optimal clinic-based pill count adherence (<90%) received additional adherence support, which included tailored counselling, monthly drug dispensing and follow-up home-visits. Participants were traced by phone and home visit if they were more than 4 weeks late in attending a scheduled clinic visit.All participants received three group counsellor-driven treatment preparedness sessions prior to commencing ART , 22. TheThe parent study was a randomised controlled trial, with primary outcome at 48 weeks, investigating the impact of mobile phone message reminders when missed doses were detected by a real-time electronic adherence monitoring device (EAMD) on adherence to ART . This deThere were five study visits: screening (week-4), baseline (week 0), weeks 16, 32 and 48, detailed in Table ®, Bayer Healthcare, Leverkusen, Germany) at baseline, 16 week and 48 week visits. Mid-dose efavirenz concentrations were determined at weeks 16 and 48. The time of blood draw, and the self-reported time of most recent ART dosing were recorded.Demographic and psychosocial details, including age, gender, weight, height as well as assessments for depression, anxiety and alcohol use, were collected at screening. The 14-question Hospital Anxiety and Depression Score (HADS) was used to assess anxiety and depression and the CAGE score used to assess alcoholism , 26. BloFor pill counts, average pharmacy refill data and self-recall, the data used was that typically available to clinic staff during a consultation with a patient.At weeks 16 and 48, study staff asked each participant: “Did you swallow your pills yesterday/2 days ago/3 days ago?” Percent doses taken over 3 days was calculated from the participants’ responses. Study staff were not part of the clinical team. Data were only available if a participant attended the study visit.Participants were instructed to bring any remaining tablets to each study visit. CPC adherence was calculated using the difference between the number of tablets dispensed and the number returned and dividing by the number of days between the date of dispensing and the current visit date (adjusted for the number of doses per day). At week 16 this would give adherence over the previous 1 month period (last dispensing visit was the SoC visit a week 12) and at week 48 adherence over the previous 2 month period (last dispensing visit was the SoC visit at week 40). For the analyses, only the tablet count related to dosing efavirenz, nevirapine or, for those who switched to second-line, lopinavir/r dosing was used.An electronic dispensing system (iDART) was used at the site to record the date of ART dispensed and the quantity given to each participant , 28. ObvThis measure used pharmacy dispensing quantities and refill visit dates to determine the number of medication-free days (days when the participant could not have had medication in hand) in each dispensing period. The number of medication-free days are subtracted from the number of days in the period, and the result divided by the number of days in the period (up to week 16 or up to week 48) to give an adherence percentage . This meAt weeks 16 and 48 visits a sample was taken to quantify mid-dosing interval efavirenz concentrations. Mid-dosing interval samples were drawn in the morning, after dosing the previous evening. Self-reported time of the evening dose was recorded. Samples were kept cold (4 °C) until transfer to the laboratory within 2–3 h of blood draw. Samples were centrifuged at 3500 revolutions/min for 10 min and plasma pipetted into cryovials which were labelled and frozen at −80 °C. Efavirenz concentrations were analysed using a validated LC–MS/MS method. The therapeutic range for mid-dose EFV concentrations is 1–4 mg/L.®) held a week of medication in an internal and removable seven compartment pill box container. Participants were trained to fill these pill boxes themselves and replace them once a week at the same time as they dosed. On opening, a signal was sent via the mobile-phone network to a secure central computer, thus recording tablet taking or treatment interruptions in real time. An adherent day was defined as any recorded EAMD opening from 06h00am on that day to 05h59am the following day. Days with battery voltage <3660 V or no heartbeat were censored (dead battery days). The daily EAMD data for each participant were downloaded from the Wisepill® server. Cumulative adherence data for EAMD was calculated as the number of adherent days divided by the number of days in care, from date of randomisation to either weeks 16 or 48.All participants received the EAMD on study entry to record daily ART dosing times. The device  at weeks 16 or 48. Nucleic acid was extracted with the NucliSens EasyMag automated extraction system . Genotyping was performed by RT-PCR followed by a nested PCR and sequencing of the complete PR gene and RT codons 1–262 (HXB2 numbering: HIV-1 nucleotide positions 2250–4229) and using gene-specific sequencing primers . ParticiData were analysed using Stata 13.0 . Descriptive statistics  were used to summarise the baseline characteristics of the participant group and to tabulate the adherence data.All available adherence data were used from each individual who had a HIV-RNA drawn within a 4 week window around week 16 (weeks 12–20) or a 16 week window around week 48 (weeks 32–64) in a per-protocol analysis from the time they entered care until the date of the respective viral load.10 values of EFV mid-dose drug concentrations were used, as log10 values shift the distribution curve of the EFV concentration values toward normal for regression modelling.Univariate and multivariable logistic regression models were used to explore each adherence measure’s relationship to virological failure and genotypic resistance at weeks 16 and 48. Both outcomes were binary, categorised as described above. The data for each adherence variable was continuous, although models were repeated using categorical variables (binary or quartiles) to assess linearity. For the TDM models, the logOther variables included in the models  were specified prior to the analysis. Receiver Operator Characteristics (ROC) were generated to view the overall predictive power of the univariate and multivariable models. The area under the curve derived from the ROC (AUC ROC), with 95% confidence intervals, was used to compare the prediction of virological failure or resistance by each adherence measure. AUC ROCs were compared using non-parametric methods.Two-hundred-thirty antiretroviral naïve HIV-positive participants were recruited onto the study between 09 July 2012 and 09 April 2013. Figure Efavirenz-based ART was initiated in 228 participants and the remaining two started twice-daily nevirapine-based ART. Both regimens were given with two background nucleoside reverse transcriptase inhibitors. Tenofovir/3TC were used in 97.8% Table . The majAt the end of the study 186 participants remained in care (80.8%) at the study site and a further 16 (7.0%) had transferred to care elsewhere. Eight participants (3.5%) had died and 19 (8.3%) were lost to follow up i.e. had not attended the clinic for more than 12 weeks and were not known to have transferred out or to have died. One participant withdrew consent.Table The subjective measure, SR, gave the highest adherence with the most individuals reporting 100% adherence . CPC, PR-gaps and PR-average also gave perfect or close to perfect adherence at week 16 and 48, but widened inter-quartile ranges highlighted some variations within the participants. Cumulative adherence by EAMD showed the lowest median adherence at both visits: 93% (IQR 74–98%) at week 16 and 86% (IQR 59–94%) at week 48. Median efavirenz concentrations were 2.3 (IQR 1.6–4.4) mg/L at week 16, and 2.1 (IQR 1.5–3.4) mg/L at week 48, within the currently recognised therapeutic range of 1.0–4.0 mg/L at both time points.At week 16, 160 participants had HIV-RNA available within the 12–20 week window and 146 (91.2%) were suppressed to ≤400 copies/ml , but the other adherence measures, including EAMD, did not significantly predict failure at week 16  participants expressed the K65R mutation-conferring resistance to tenofovir, and three the M184 V mutation- conferring resistance to lamivudine/emtricitabine. All 10 who expressed resistance had NNRTI mutations, including L100I, K101E, K103N, V106M, Y181C, Y188C/Y/L, G190A/G/S and H221HY. Despite only efavirenz and nevirapine being used, mutations conferring resistance to etravirine and rilpivirine were noted in three participants: one each of V90I, E138A and V179D.Twenty-seven participants had a HIV RNA of >40 copies/ml at week 48. Eleven of the fourteen (51.9%) who had HIV RNA >500 copies/ml had resistance genotyping successfully completed. One had no major resistance mutations. Two (18%) participants expressed the K65R mutation and four (36%) the M184 V mutation. As at week 16, the majority of the remaining mutations, in all 11 who expressed resistance, were NNRTI mutations, including L100I, K101E, K103N, V106M, Y181C, G190A, H221HY and F227L. Again, mutations to etravirine and rilpivirine were noted in 3 participants: one with E138A and two with V179D.10 EFV concentration were all significantly predictive of resistance in both univariate and multivariable analyses at week 48  Table . ReducedWe found high levels of adherence using both short-term and cumulative measures. Three-day SR yielded the highest adherence estimate, but was not a significant predictor of either viral suppression or resistance. This is consistent with findings which show that while short-term SR measures are widely utilised and useful for clinical intervention , they ovIn contrast, four of the five objective adherence measures effectively predicted virological failure: CPC, PR-average, PR-gaps and EAMD data. Cumulative PR measures (PR-average and PR-gap) were among the best predictors of virological failure and resistance at 48 and, of note, PR-average was the only predictor of resistance at week 16. PR-average has previously been reported to be a reliable predictor of virological outcomes and mortality , 8, 9, wCPC is not widely recommended as it is subject to “pill dumping” and can be complex to perform in a large clinic . NonetheWhile EAMD data were among the best at predicting outcome in this study at week 48, EAMDs are not routinely used in clinical care. However, recent data shows EAMD can reduce costs associated with HIV-RNA monitoring and real-time devices can detect early virologic rebound before established failure , 38, 39.By altering the distribution of efavirenz concentrations through the use of log values in the regression model, we found that the log values of mid-dose EFV concentration were predictive of resistance at week 48, with wide confidence intervals, but not of failure. Most drug concentrations were therapeutic, possibly reflecting white coat pre-visit dosing, leaving few with concentrations below therapeutic where virological failure would be more likely. The few participants with sub-therapeutic concentrations limit the interpretation of this data. Larger studies including more participants with sub-therapeutic EFV concentrations are needed to adequately explore the ability of TDM to predict virological failure and resistance.Most participants remaining in care at weeks 16 and 48 had virological suppression. The majority of those who had detectable HIV RNA also had resistance that would compromise at least one drug in their antiretroviral regimen, even at 16 weeks. Only the PR-average method predicted this early resistance. Using early pharmacy refill data to predict failure is practical and easy to achieve both for an individual and on a programmatic level. EAMD data was highly predictive of resistance at week 48, as were both PR measures and, to a lesser extent CPC.It is interesting that while many of the adherence measures are predictive of viral or resistance outcomes at week 48, far fewer are predictive at week 16. This might suggest that measures of adherence which cover a longer period of dosing are more accurate e.g. cumulative EAMD, PR methods and CPC over 2-months. Conversely, adherence data collected over a shorter window were not predictive e.g. 3-day self-report, spot EFV concentrations and CPC over 1 month.The prospective collection of multiple adherence measures in a single cohort is the strength of our study as it has allowed direct comparison of these measures—a comparison rarely achieved , 16. OurBoth genotyping and viral load testing are costly and funding to conduct baseline resistance testing and confirmatory viral loads at week 48 was not available. This is a study limitation. In addition, SR data was only collected as a 3-day recall at each visit and CPC data were only available for the 1 or 2 months preceding the week 16 or 48 visit and not over the complete study period. These measures may have performed better being collected over a longer or cumulative period of time.Adherence as measured by CPC, PR measures and EAMD were the best predictors of resistance and virological failure in this prospective study. Pill counting can be implemented at any clinic, and pharmacy refill data is already widely available and an immediately implementable option, particularly in resource-poor settings. Consideration should also be given to the use of electronic measures as adherence monitoring strategies as costs reduce .Additional file 1. Supplementary tables.Additional file 2. Study data on which manuscript analyses are based.


Antiretroviral treatment (ART) has been massively scaled up to decrease human immunodeficiency virus (HIV)–related morbidity, mortality, and HIV transmission. However, despite documented increases in ART coverage, morbidity and mortality have remained substantial. This study describes trends in the numbers and characteristics of patients with very advanced HIV disease in the Western Cape, South Africa.Annual cross-sectional snapshots of CD4 distributions were described over 10 years, derived from a province-wide cohort of all HIV patients receiving CD4 cell count testing in the public sector. Patients with a first CD4 count <50 cells/µL in each year were characterized with respect to prior CD4 and viral load testing, ART access, and retention in ART care.Patients attending HIV care for the first time initially constituted the largest group of those with CD4 count <50 cells/µL, dropping proportionally over the decade from 60.9% to 26.7%. By contrast, the proportion who were ART experienced increased from 14.3% to 56.7%. In patients with CD4 counts <50 cells/µL in 2016, 51.8% were ART experienced, of whom 76% could be confirmed to be off ART or had recent viremia. More than half who were ART experienced with a CD4 count <50 cells/µL in 2016 were men, compared to approximately one-third of all patients on ART in the same year.Ongoing HIV-associated morbidity now results largely from treatment-experienced patients not being in continuous care or not being fully virologically suppressed. Innovative interventions to retain ART patients in effective care are an essential priority for the ongoing HIV response. The ini for ART , 4, culm for ART , 6. Sout for ART . As the  for ART .Approximately 420000 people out of a total population of 6.3 million were projected to be living with HIV in the Western Cape province of South Africa in 2016. Approximately half of all HIV-infected individuals and >85% of those with a CD4 count <200 cells/µL were estimated to be on ART . The curThis study describes temporal trends in the ascertainment of very advanced HIV disease in the Western Cape province of South Africa, evidenced by CD4 counts <50 cells/µL, and trends in the HIV treatment history of those with very advanced HIV disease.The Western Cape is one of 9 provinces in South Africa. One in 5 pregnant women has HIV. The vast majority of people living with HIV seek care in the public sector, which first offered ART in 2001, accelerating coverage after 2004 when ART provision became national policy . CD4 monAll public sector laboratory testing is done by the National Health Laboratory Service (NHLS) and the digitized results have been available to the province since 2007. The province has successfully established a patient registration system which shares a unique health identifier and Patient Master Index (PMI) across both hospital and ambulatory services, which reached near-complete coverage as of 2013 . This haAnnual cross-sectional snapshots of CD4 distributions were described from a derived province-wide HIV cohort of all patients receiving CD4 testing. Further longitudinal analyses focused on CD4 testing and ART treatment history in adults (≥16 years old) who were identified with CD4 <50 cells/µL in each period.The cohort is based on data linkage between the laboratory-reported CD4 and viral load tests, and HIV treatment information systems. Data which had been previously linked for HIV operational reporting purposes were used for analyses until the end of 2012. Analyses for subsequent years were based on anonymized extracts from the PHDC. In this period, 98.7% of CD4 counts could be linked to the PMI.In the manually linked database, the CD4 and viral load data had been linked to each other and to the HIV treatment database, using a combination of probabilistic and deterministic linkage based on name, surname, date of birth, sex, facility, subdistrict, district, and clinic folder number. Fine Grain Record Linkage (FRIL) version 2.1.5 (Emory University) and Stata version 13.0 (StataCorp) software were used for linkage and analysis. A random sample of linkages was manually validated. These data were anonymized prior to use for this analysis. Data from both sources combined were available from 2007 until 20 September 2017, and for 2017, all counts were upweighted to represent a full year’s worth of data to retain comparability with previous years.The data are presented in annual cohorts based on the year the CD4 count was obtained. Results from 2007 are omitted as prior CD4 count testing could not be determined. Province-wide CD4 counts are described as absolute counts of tests performed as well as by counts of unique individual patients receiving testing in each year. Unique patients per year with CD4 <50 cells/µL were further stratified into the following categories in relation to ART status: late presenters (presenting for their first ever CD4 test with CD4 <50 cells/µL); previously not eligible for ART (inadequate pre-ART care); previously eligible for ART (failed linkage to or initiation of ART care); and those with a low CD4 count after ART initiation. ART initiation was determined by an ART start date recorded in the provincial electronic HIV registers or a viral load result, in which case the ART treatment start date was estimated as 4 months prior to the first recorded viral load . Patients were deemed eligible for ART if their CD4 was <200 cells/µL prior to 1 May 2010 or <350 cells/µL after 1 May 2010 as per operative national guidelines.ART-experienced patients in whom visit-level treatment records were available were further stratified into 4 categories: lost to follow-up  prior to their low CD4 count, including those who returned to care within 30 days prior to the low CD4 count but otherwise met this definition; those in care prior to the low CD4 count but who had had a recent (ending in the previous year) gap of >90 days without antiretroviral medicines; or continuously in care . Patients without medicine information linked to their visit were assumed to have been given 30 days of treatment and therefore were counted as lost to care at 120 days after their last appointment.Within those in care (with or without gaps), patients were further stratified by those without viral load data, those with suppressed viral loads, those with viremia , and those failing treatment .P values . Age, duration on ART, and time between tests were described as medians and interquartile ranges.Results of hypothesis testing for highlighted differences in proportions or tests for trend are not presented due to the large numbers of patients involved and universally low The study was approved by the University of Cape Town Human Research Ethics Committee (421/2016).Over the 10-year period from 2008 through 2017, the total number of patients estimated to be living with HIV in the Western Cape increased to >400000, with the proportion estimated to be on ART increasing from 16% in 2008 to 52% in 2016 . CD4 couThe number of unique patients per year with a lowest CD4 count test result <50 cells/µL varied between 6164 and 8133, remaining relatively constant over time . These pPatients presenting for the first time initially constituted the largest group of those with very low CD4 counts, dropping proportionally over time from 60.9% to 26.7% . By contOf those ART naive with previous CD4 count testing prior to the index CD4 test (first CD4 <50 cells/µL per patient per year), the median time from the previous value to the index CD4 count increased over time. The time between consecutive CD4 counts was much longer in those who had previously been ART ineligible than those who had been eligible for ART , and less likely to be virologically suppressed if tested (9.7% vs 19.5% of those tested in 2016). A higher number of those on ART with viremia (>1000 copies) had evidence of sustained viremia over ≥2 or more (56% in 2016).Looking across all patients with CD4 counts <50 cells/µL in 2016, 51.8% were ART experienced, among whom 76% could be confirmed to be off ART or had viremia at the time of the index CD4 test and 7% could be confirmed to be on suppressive ART; the virologic status of the remaining 17% was not known . The totThis analysis has demonstrated how in a high-burden HIV setting, the proportion of patients presenting with very advanced HIV disease is not declining despite the successful scaling up of ART. This is reflected clinically by the relatively stable number of cases of cryptococcosis during the last decade, despite a considerable increase in access to ART. The proportion of patients initially presenting with very advanced HIV disease is declining, reflecting increased access to treatment; however, this reduction is being offset by an increasing number of patients who have previously started ART and are re-presenting with advanced HIV following a period without effective ART. Most often this is due to predictable reasons such as stopping ART, poor adherence, or virological failure, many of which are amenable to intervention.The initial interest from hospital-based clinicians in the province in the pattern of presentation with very low CD4 counts was in response to ongoing morbidity from conditions such as cryptococcal meningitis, which are associated with advanced immunodeficiency. Clinician-triggered and, more recently, laboratory-triggered reflex screening for cryptococcal antigenemia was introduced in 2014 and 2016, respectively, for all patients with CD4 counts <100 cells/µL. Although this likely prevents cryptococcal meningitis in some patients , there hThe 90-90-90 strategy has successfully focused energy on identifying patients with HIV and linking them to care, bolstered by “Treat-all” guidelines. Many organizations have, as a result, concentrated in recent years on the identification of those with undiagnosed HIV and on improved linkage to care. The declining number of patients presenting for the first time in this study with advanced HIV disease points to the success of expanded access to HIV testing, care, and treatment. In many settings, the 90% target for diagnosing HIV in those infected is close to achievement . The domOur findings point to extensive missed opportunities for intervention in patients who had started ART. The majority had either fallen out of care where reengagement interventions could have been triggered by missed appointments or formally defined LTFU, or had previously had viremia or had missed viral load testing. Of those with viremia, half had been confirmed to be so on >1 consecutive occasion. Clinical governance and quality-improvement processes could address the efficient implementation of guidelines for virologic testing, and adherence promotion and regimen switching in patients fulfilling virological failure criteria. The increasing affordability and tolerability of new regimens should remove concerns around premature switching, and the balance should shift to more aggressive intervention to ensure suppressive ART. The study also corroborated previous findings of excess mortality risks in men  which, a“Community viral load” tracks transmission potential , 25, butThere are several limitations to this study. Two separate data linkage exercises were stitched together to create a 10-year trend history, and while they aligned remarkably well at the seam, it would have been preferable to have a single approach. There may have been residual failures to link observations due to incomplete data for patient matching, either missing antecedent CD4 count histories or subsequent ART initiation. In terms of the major findings from this study, however, both biases, if present, would result in underestimation rather than overestimation of the trends toward fewer presentations in newly diagnosed patients and more in care- or treatment-experienced patients. Some of the trends may further be artefact of the availability of data only from 2007 onward, especially durations, as previous measures, by design, should get longer with increasing calendar time due to the lack of prior data in the early years of the analysis.This study has demonstrated that ongoing HIV-associated morbidity now results largely from treatment-experienced patients not being in continuous care or not maintaining virologic suppression. Attention of healthcare services will need in the future to focus much more aggressively on the innovation and investment in and quality of ART services in order to avert transmission, morbidity, and mortality. In this study we have shown that monitoring the absolute numbers of very low CD4 counts at a population level over 10 years provided a readily accessible, durable, and sensitive integrated indicator of overall program performance and identified specific program deficiencies.


While antiretroviral therapy (ART) availability for HIV patients has increased dramatically in Uganda, patient linkage to and retention in care remains a challenge. We assessed patterns of engagement in care in 20 Ugandan health facilities with low retention based on national reporting.We assessed patient linkage to care (defined as registering for pre-ART or ART care at the facility within 1 month of HIV diagnosis) and 6-month retention in care (having a visit 3-6 months after ART initiation) and associations with patient−/facility-level factors using multivariate logistic regression.s = 0.06; p = 0.82). Females, adolescents, and patients in rural settings tended to have lower linkage and retention in multivariable-adjusted models.Among 928 newly HIV-diagnosed patients, only 53.0% linked to care within 1 month. Of these, 83.7% linked within 1 week. Among 678 newly initiated ART patients, 14.5% never returned for a follow-up visit at the facility. Retention was 71.7% according to our primary definition but much lower if stricter definitions were used. Most patients were already falling behind appointment schedules at their first ART follow-up (median: 28 days post-initiation vs. recommended 14 days). 27.3% of newly-initiated patients had follow-up appointments scheduled 45+ days apart rather than monthly per national guidelines. Linkage and retention were not strongly correlated with each other within facilities .The online version of this article (10.1186/s12879-018-3042-8) contains supplementary material, which is available to authorized users. Antiretroviral therapy (ART) coverage for HIV patients has been increasing steadily in Uganda. Of the estimated 1.5 million people living with HIV in Uganda in 2015,  and otheHigh attrition rates pose a serious concern to HIV programs as treatment discontinuation leads to increased viral load and associated increases in morbidity, mortality, incidence of drug resistance and risk of transmission, ultimately undermining the benefits achieved from implementing treatment programs. Additionally, high attrition will require costly efforts to re-find and re-initiate patients to meet and sustain Uganda’s ART scale-up targets. Thus, there is an increasing need for focused attention on linkage to and retention in care to improve patient outcomes and successfully control Uganda’s HIV epidemic.A number of groups have studied barriers to linkage to and retention in ART care in Uganda and neighboring countries. Barriers reported by patients included transportation costs, long wait times, and stigma . In one More evidence is needed on the patterns around and predictors of patient engagement in care in order to generate evidence on scalable and sustainable interventions for improvement. We retrospectively assessed patient linkage to and retention in care and service delivery barriers among 20 health facilities in Central Uganda. While this group of facilities is not nationally representative, the findings from this analysis can help to identify patterns in poorly performing facilities and opportunities for health systems strengthening.n = 4) were excluded for logistical reasons and one specialized mental health center was excluded.Twenty facilities with low retention were randomly selected across 14 districts in Central Uganda  to participate in a study involving an intervention to promote proactive follow-up and counseling practices. Facility eligibility criteria included offering ART to pediatric and adult patients starting January 1, 2015 or earlier, demonstrating a low estimated annual retention among patients on ART  2 estimates), and having a high ART patient volume (> 120 patients enrolled in 2015 according to DHIS 2). All eligible facilities were level III or IV, medium-sized health facilities offering general services but no specialized services. Eligible facilities located on islands  on tablets.Patients of all ages who tested HIV-positive at study facilities between December 25, 2015 and June 25, 2016 were sampled for inclusion in the linkage assessment. Based on preliminary power calculations, data collectors systematically sampled HIV-positive patients in the HIV Testing and Counselling Register such that a minimum of 40 patients were sampled per facility and the smallest possible number of patients beyond that were sampled. However, all pediatric patients were sampled. Also, if the number of patients testing during the relevant time period was less than 60 patients, all patients were sampled. Pre-ART and ART registers were assessed in the 3 months after diagnosis to confirm which HIV-positive patients linked to care at the facility. Due to logistical and budgetary constraints, it was not feasible to follow up at other facilities to search for patients who did not link to care at the facility where the test was conducted.The primary definition of linkage to care was defined as registering for pre-ART or ART care at the facility within 1 month (30 days) of HIV diagnosis, as the WHO recommends timely linkage to care to ensure that patients access appropriate treatment as soon as possible , 16. PatAll patients  who initiated ART at study facilities between December 25, 2015 and March 25, 2016 and were listed on the ART register were included in the retention assessment. Data on patient demographics and scheduled and attended ART appointments through 6 months post-initiation were extracted from patient care cards.Our a priori primary definition of 6-month retention was attending at least 6 ART appointments in the first 6 months after ART initiation. This represents full adherence to the ART visit schedule in the National ART Guidelines as of 2016, in which patients should attend an ART appointment 2 weeks after ART initiation and monthly thereafter for the first year after initiation. Revised guidelines in 2017 required appointments at 2 weeks, monthly for 3 months, and quarterly thereafter. We also assessed six-month retention according to the Uganda MOH definition: Having a visit within the previous quarter (3-6 months after ART initiation). To understand patient appointment spacing, we calculated median number of patient appointments over 6 months and compared appointment spacing to national guidelines.This analysis is from the baseline assessment and includes pre-intervention data from December 2015-September 2016.Proportions and medians/interquartile ranges (IQR) were calculated to describe program and patient characteristics. Predictors of linkage and retention were assessed using logistic regression models using cluster-robust standard errors given facility-level data clustering. The missing indicator method was used to account for missing data . VariablDetailed description of the patient follow-up practices at study facilities and challenges identified can be found in Supplemental Digital Content  and was poorest in adolescents (ages 10-18 years) compared to adults (ages 19-48 years) . In particular, adolescent females had the lowest linkage (35.5%) of any sex/age category combination. In addition, in univariate models, significantly lower linkage to care was seen in rural/remote facilities compared to semi−/peri-urban facilities . No differences were seen by facility level, the number of days per week that ART was offered at the facility, or the number of expert clients at the facility. We also assessed other facility-level characteristics such as year of start of implementation of ART program, and reports of stockouts, but these variables were not associated with linkage to care. In multivariable models, significantly lower linkage was seen in adolescents and in patients in rural settings.In univariate models, linkage was slightly lower in women than men, although the difference was not statistically significant  and was very low in adolescents versus adults 19-48 years . Lower retention was observed in rural/remote environments compared to semi−/peri-urban , and in facilities with fewer expert clients to provide counseling and follow up with patients who missed appointments . Facilities offering ART appointments on fewer days  appeared to have slightly lower retention, although this was not statistically significant. No differences in patient retention were seen by facility level, date of ART program initiation at the facility, or other facility-level characteristics. When all statistically significant characteristics were included in a multivariable model, only female sex and rural location remained statistically significant predictors of low retention.In univariate models, retention was slightly lower in women compared to men . The interclass correlation coefficient (ICC/rho) was 0.047 for linkage and 0.025 for retention. Ninety-eight patients who were found to link to care in the linkage dataset also appeared in the retention dataset. Of those, patients who linked to care within the first week of HIV diagnosis appeared to be more likely to be retained in care compared to patients who linked after 1 week, although with limited statistical power in this analysis, the difference was not statistically significant .There was substantial variability in linkage and retention by facility and district. Linkage and retention were not strongly correlated with each other within facilities (rThe median (IQR) time between ART initiation and first follow-up appointment was 28 days (27-42) per person, compared to 14 days as per national guidelines. 27.3% of newly initiated patients had follow-up appointments scheduled at least 45 days apart. This meant that the median (IQR) time between subsequent appointments was 39 (28-56) days per person, versus 28 days as per national guidelines.Linkage and retention were low  at the 20 study facilities in Central Uganda and tended to be lower in women, adolescents, and patients living in rural settings. Lower retention was seen in facilities with 1 or fewer expert clients to follow up with patients who missed appointments compared to 5 or more expert clients, although this association did not remain statistically significant in multivariable models. Of note, most patients who tested positive for HIV and subsequently linked to care did so within 1 week of diagnosis, indicating that linkage support may be most critical in the first week after testing positive. There was substantial variability in linkage and retention between facilities, and linkage was not strongly correlated with retention. Numerous facility-level factors were identified that may have contributed to low linkage and retention, such as poor recordkeeping systems, inadequate funding for patient phone follow-up and home visits, and limited staff. In addition, poor staff supervision and motivation appeared to be an issue at some facilities. Finally, we observed that a substantial proportion of patients appeared to be receiving multi-month appointments in the first months after ART initiation, contrary to current national guidelines.The low linkage and retention at facilities in this study were not surprising given that these facilities were selected based on suspected poor patient retention based on national reporting and justify increased focus on strengthening health systems at facilities like these. Facility-level inefficiencies and constraints identified by facility staff such as limited funding and staff capacity for phone follow-up and home visits to patients who miss appointments, lack of private space for patient counseling, and stockouts echo common barriers to care in sub-Saharan Africa that have been previously described in the literature –22. FactAmong patients who linked to care within 3 months, most (83.6%) linked within 1 week, suggesting that linkage support may be critical in the first weeks after patients test positive. Patients who linked to care within 1 week appeared to be slightly more likely to be retained in care, although this difference was not statistically significant. Similarly, in the US National Surveillance system, among 20,572 people diagnosed with HIV and linking within 3 months, 81.7% linked within the first month (linkage within 1 week was not assessed) and the percentage of patients achieving viral suppression was higher among patients who linked more quickly . In addiThe data from this study suggests that some facilities may be scheduling multi-month visits for newly initiated patients, rather than more frequent visits according to national guidelines. This observation should be assessed further, particularly in light of new efforts to promote differentiated models of care for different types of patients. At present, differentiated care usually is meant to begin after 6-12 months of ART. Also, the median time to first follow-up was 28 days which may indicate a change in regimens away from nevirapine-based regimens which require a lead-in dose; it could be dangerous for patients on these regimens to wait more than 2 weeks for an appointment. In the future, it will be important to better understand the factors driving the timing of appointments by clinicians and implications on patient retention. There is need to understand why nearly 15% of clients never returned for a second appointment. This could have important long-term implications as the national program rolls out its Test and Start strategy, especially if readiness for ART is not assessed appropriately before initiating ART.This study is limited by its reliance on facility staff reporting and retrospective data from health records. Because data collection was limited to the 20 facilities in our study, we could not confirm outcomes of patients who were LTFU at the facility, meaning that some of the patients who appeared to be LTFU may in fact be in care elsewhere and our estimates of linkage and retention are likely to be underestimates. Nevertheless, our findings highlight relatively low levels of linkage and retention such that even if the percentages of patients LTFU are overestimates, they remain alarmingly high. Due to funding limitations, we were only able to follow up patients to assess retention after 6 months rather than 1 year or more as reported in some other studies . FinallyBeyond understanding patterns and predictors of linkage and retention, the next step will be designing and implementing effective interventions to improve engagement in care. Although the number and quality of studies is limited, several interventions have been identified that may be effective. Home-based HIV testing and counseling with follow-up support and community-based testing appear to be promising strategies to increase linkage , 35 and Looking to the future, a number of new programs and policies may impact patient engagement in care. New Test and Start policies could reduce retention because asymptomatic patients will be put on treatment and may be less likely to stay in care due to the side effects they experience on treatment. However, better drug regimens that will soon be standard first line regimens such as Dolutegravir may play an important role in improving retention because of increased tolerability and reduced side effects –44. ThroThis study suggests that patients who test HIV-positive may benefit from immediate support in order to link to care in a timely manner. There should be additional focus on adolescents to improve linkage to care and retention in care in this age group. Further investigation is needed on clinician appointment scheduling practices and implications on retention.Additional file 1:Supplementary Results. This section provides additional facility-level information on existing barriers to patient follow-up, counseling, linkage to care, and retention in care. (DOCX 116 kb)Additional file 2:Table S1. This table provides the percentage of individuals linked to care and retained in care according to different definitions of linkage and retention, as well as the median (IQR) days from HIV diagnosis to linkage among patients who did link to care. (DOCX 14 kb)


The aim of this study was to determine the effects of nutritional status at the start of highly active anti-retroviral therapy on treatment outcomes among HIV/AIDS patients taking HAART at Jimma University Specialized Hospital.We performed a retrospective cohort study involving 340 adults who started highly active anti-retroviral therapy. The patients have been clinically followed for 2 years. Data were extracted from paper based medical charts by trained data collectors from January 30 to February 28, 2014 using data collection format. We entered data into Epi data version 3.1 and then exported to SPSS for windows version 21. Predictors of CD4 change were identified using multivariable linear regression model. Time to an event (death) was estimated by Kaplan–Meier and predictors of mortality were identified by Cox proportional hazard model.Out of 340 patients, 42 patients died during the follow-up. Twenty-five (59.5 %) deaths were from malnourished group. Age, baseline CD4, sex, baseline HAART and marital status were significant predictors of immunologic recovery at different time points. Malnutrition was associated with lower CD4 recovery and greater hazard of death.Malnutrition tends to decrease CD4 recovery and predisposes patient to early death. Treatment of HIV-infected patients with highly active antiretroviral therapy (ART) leads to immune reconstitution as shown by increases in CD4 lymphocyte counts, decreased risk of opportunistic infections and improved survival , 2. HoweEven though, previous studies showed malnutrition was independent predictor of death in patients taking HAART –8 in difHowever, no previous study had holistically examined the impact on survival, CD4 recovery and occurrence of opportunistic infections of malnutrition at the time of starting HAART. Furthermore, there were few studies in Africa and no study done in Ethiopia that examined effect of malnutrition at the initiation of HAART on treatment outcome. It is possible that malnutrition may impair the immune response to HAART, prolong the period during which patients are at risk of opportunistic infection and directly or indirectly increasing the risk of death. Malnutrition may therefore, represent a potentially reversible cause of increased mortality in patients who are initiating ART.We conducted retrospective cohort study at Jimma University Specialized Hospital, the only teaching and referral hospital with bed capacity of 450 in the South Western part of the country providing specialized health service for approximately 9000 inpatients and 80,000 outpatients each. The ART clinic of the hospital started providing service to people living with HIV/AIDS (PLWA) in 2002. Since establishment the clinic had 3700 patients following care and treatment . The priThe data was cleaned, edited and completed using Epi data version 3.1 and exported to SPSS for window version 21 for analysis. The main analysis in this study was linear regression for CD4 recovery and survival analysis for death. Variables with p value of <0.25 in bivariate analysis and BMI were fitted to multivariable cox-proportional analysis model to identify independent predictors of CD4 change at all time points. Kaplan–Meier survival analysis was done to estimate the survival time. Log-rank test was used to compare the KM curves for two or more categories of patients on HAART. Predictors of event (death) over a period of time t, was analyzed by Cox proportional hazard model. The level of significance was set at p value less than 0.05 and 95 % confidence intervals (CI) were used throughout. Multi-collinerity was checked for independent variables before fitting into multivariable analysis.2. The median baseline CD4 count was 144.5 [IQR 89–209] cells/mm3  deaths were from malnourished group. The mean age of the study participants was 34 [IQR 26–38] years. The median age was 30 years and 200 (58.8 %) patients were females. Mean baseline BMI was 1.50 [IQR 1.0–2.00] kg/m3 (p = 0.003). CD4 count of malnourished group decreases by 12.40 cells/mm3 (p = 0.500) compared to well-nourished group . CD4 count of patients with baseline CD4 count in the range of 100–199 increases by 201.29 cells/mm3 (p = 0.047) compared to patients with baseline CD4 count greater than 350. CD4 count of malnourished patients decreases by 21.5 cells/mm3 (p = 0.321) compared with well-nourished group at 12 months (Table Age and baseline CD4 count were independent predictors of immunologic recovery. For 1 year increase in the age of the patient CD4 decreases by 1.91 cells/mm3 (p = 0.005). Females had increased CD4 count by 104.3 cells/mm3 compared to males. Married patients had 138.56 cells/mm3 (p = 0.005) higher CD4 count compared to widowed patients. CD4 count of patients who started treatment with Stavudine based regimen increases by 87.49 cells/mm3 (p = 0.036) compared to CD4 count of patients who started with Tenofovir based regimen. Malnourished group has 22.4 cells/mm3 lower increases in CD4 count compared to well-nourished group , WHO clinical stage (p = 0.022), baseline HAART (p = 0.010) and Fluconazole prophylaxis (p = 0.000). Even though, malnourished patients had shorter survival times compared to well-nourished group, it was not statistically significant (p = 0.170) , Tenofovir , Fluconazole prophylaxis , age above 50 years  and CD4 greater than 200 . Malnourished patients were 1.3 times at risk of death compared to well-nourished group  (Table There was no significant difference between malnourished and well-nourished patients in terms of CD4 recovery after HAART initiation. This finding is similar with reports by Elizabeth et al. who reported nutritional status at the start of HAART as measured by BMI, FFMI, FMI and skin folds did not predict good post HAART CD4 change at 6, 12 and 24 months in a cohort of HIV infected Rwanda women . Lack of3 at baseline was associated with smaller increase in CD4 at 6 and 12 months in multivariable model compared to patients with lower CD4 counts. This finding was also documented in London, UK where lower CD4 count was associated with greater increase in CD4 after 3 month of HAART initiation both in multivariable model  times at greater risk of death compared to those with BMI >18.5 kg/m2 [2 [Malnourished patients were almost two times more likely to die early compared to well-nourished group though it is not significant in Multivariable Cox proportional model. This finding was in line with study done in Zewditu Memorial hospital, Ethiopia which shows patients with BMI of <18.5 kg/m.5 kg/m2 . This fikg/m2 [2 . Studieskg/m2 [2 , 23. Likkg/m2 [2  also repTenofovir based regimen was associated with highest risk of death compared to Stavudine and Zidovudine based regimen. This is similar with what Kelechi and his colleagues reported  but in cIndependent of specific antiretrovirals, Fluconazole prophylaxis was found to be strong predictor of death in our study and indifference to a Uganda study suggesting that this antifungal did not impact mortality under similar conditions . Most paBMI at the start did not predict change in CD4 at any time point after initiation of HAART after adjustment for other variables.Age of the patients was significant predictor of immunologic outcome at 6, 12, 24 months adjusting for other factors. Baseline CD4 count was significant predictor of CD4 change at 12 months. Sex was significant predictor of immunologic outcome at 24 months after HAART initiation. At 24th month baseline HAART and marital status predicts immunologic outcome.Age >50 years of age, Tenofovir based regimen, Zidovudine based regimen, taking fluconazole and CD4 <200 were associated with greater risk of death.Malnutrition at the start of HAART does not predict early death of patients.


By assessing the changes in concentration of soluble receptor activator of nuclear factor κ B ligand (RANKL) and osteoprotegrin (OPG) after initiation of combination antiretroviral therapy (cART) in treatment-naïve HIV-infected patients we aimed to evaluate whether the initial accelerated bone loss could be mediated by increased soluble RANKL (sRANKL) levels associated with CD4+ T cell recovery.We used multiplex immunoassays to determine sRANKL and OPG concentrations in plasma from 48 HIV patients at baseline and 12, 24, 48 and 96 weeks after cART initiation.p = 0.02) with 25% decrease (95% CI: -42 to −5) at week 24 compared to baseline and stabilized at a lower level thereafter. We found no correlation between CD4+ T cell count increment and changes in sRANKL or between percentage change in BMD and changes in sRANKL.Soluble RANKL changed significantly over time (overall In this study there was no indication that the accelerated bone loss after cART initiation was mediated by early changes in sRANKL due to CD4+ T cell recovery. Future studies should focus on the initial weeks after initiation of cART.Clinical-Trial.gov. id NCT00135460, August 25, 2005. The study was approved by the Danish Data Protection Agency, Danish Medicines Agency and Regional Ethics Committee. HIV-infected patients experience a loss in bone mineral density (BMD) during the first 6–12 months after initiation of combination antiretroviral therapy (cART) . The bonA key system in bone turnover is the RANK/RANKL/OPG system . Recepto3 . These diverting results may be attributable to several differences in study design including study population size (n = 48 vs n = 19), difference in ethnic origin and a TDF-containing regimen in the study by Ofotokun et al. We also had a lower proportion of patients with very low CD4 cell counts. In our study, only five patients (10%) had baseline CD4 + T cell count below 50 cells/μl, which may have hampered the power to find an association between CD4+ T cell reconstitution, changes in sRANKL and changes in BMD.Despite animal models having shown a positive correlation between increasing CD4 + T cell count and sRANKL and BMD loss , we did including measurements at week 12 and 24, the period where the accelerated bone loss induced by cART initiation peaks. Only one other group has successfully measured sRANKL levels at multiple time points within 24 weeks of cART initiation [The major strength of this prospective study is that we repeatedly measured sRANKL within the two first years after cART initiation itiation . In our Our study has several limitations. The study population is relatively small and there is a risk of type II error given the large biological variability of sRANKL . We did In summary, we found that sRANKL decreased to levels below baseline values from week 24 and onwards after initiation of cART. In our study, in which median baseline CD4 + T cell count was around 200 cells/μl, there was no indication that the early cART induced accelerated bone loss was mediated through increased levels of sRANKL induced by CD4+ T cell recovery. Further studies focusing on the initial weeks after initiation of cART are warranted.


CRF19_cpx is a complex circulating recombination form (CRF) of HIV-1. We describe the characteristics of an outbreak of the CRF19_cpx variant among treatment-naïve patients in southern Spain.The study was undertaken at the Virgen de la Victoria Hospital, a reference centre for the analysis of HIV-1 genotype in Malaga (Spain). Subtyping was performed through REGA v3.0 and the relationship of our CRF19_cpx sequences, among themselves and regarding other reference sequences from the same variant, was defined by phylogenetic analysis. We used PhyML program to perform a reconstruction of the phylogeny by Maximum Likelihood method as well as further confirmation of the transmission clusters by Bayesian inference. Additionally, we collected demographic, clinical and immunovirological data.Between 2011 and 2016, we detected 57 treatment-naïve patients with the CRF19_cpx variant. Of these, 55 conformed a very well-defined transmission cluster, phylogenetically close to CRF19_cpx sequences from the United Kingdom. The origin of this subtype in Malaga was dated between 2007 and 2010. Over 50% of the patients presented the non-nucleoside reverse transcriptase inhibitor G190A resistance mutation. This variant was mostly represented by young adult Spanish men who had sex with men. Almost half of them were recent seroconverters, though a similar percentage was diagnosed at a late state of HIV infection. Five cases of AIDS and one non-AIDS defined death occurred during follow-up. The majority of patients treated with first-line combination antiretroviral therapy (ART) responded.We report the largest HIV-1 CRF19_cpx cohort of treatment-naïve patients outside Cuba, almost all emerging as an outbreak in the South of Spain. Half the cases had the G190A resistance mutation. Unlike previous studies, the variant from Malaga seems less pathogenic, with few AIDS events and an excellent response to ART. Although molecular epidemiology studies suggest a central African origin for this variant , CRF19_cClinically, patients with the subtype CRF19_cpx are mostly linked to a rapid progression to AIDS . CRF19_cAs mentioned, and despite the very low spread of the CRF19_cpx variant outside Cuba, the existence of a few cases of this recombinant form was recently reported in Tunisia and Spain –11. The Here, we present a major expansion of this HIV-1 CRF outside Cuba. The variant has been transmitted in southern Spain as an outbreak among MSM, diagnosed between 2011 and 2016.pol gene, encoding the complete protease (PR) and partial reverse transcriptase (RT), was sequenced using RT–PCR and Sanger sequencing  or 454 pyrosequencing , depending on the date of sample collection . The subtype for each FASTA sequence provided was assigned through REGA v3.0 and sequences determined as CRF19_cpx subtype, afterwards confirmed by phylogenetic analysis.The study was undertaken at the Virgen de la Victoria Hospital, a reference centre of the study of HIV-1 genotype drug resistance for six hospitals from the region of Malaga (southern Spain). A genotype resistance test has been routinely undertaken in our centre since 2004 for all the patients with confirmed HIV-1 infection at the time of diagnosis and before starting combination antiretroviral therapy (ART). A partial region of HIV-1 The relationship of our CRF19 cpx sequences among themselves and with regards to the epidemic of this subtype worldwide was characterized by means of a phylogenetic analysis with another 254 reference sequences of the same variant retrieved from the LANL . The PR and RT sequences  were aligned by ClustalX. The phylogenetic reconstruction was inferred by maximum likelihood method with PhyML v3.0 program –13. The http://tree.bio.ed.ac.uk/software/figtree/).The phylogeny inferred was confirmed by a Bayesian approach using MrBayes v3.2 program  and visu-3 substitutions per site per year (standard deviation = 0.5) in the evolution rate, as the general heterogeneity rate reported in the literature for all subtypes in the pol gene  (results in S1 Table). The highest prevalence for this subtype was found in 2016, with 19 patients out of 438 (4.3%).We subtyped all sequences provided by any kind of genotype test performed in our hospital from treatment-naïve patients diagnosed since 2004, finding the first case of the CRF19_cpx variant in 2011 . We inferred the phylogeny with the GTR model using a Gamma distribution (+G) with 5 categories and assuming a fraction of sites as evolutionarily invariant positions (+I), according to results from FindModel . Almost all of the sequences studied (55 out of 57) appeared phylogenetically close  to another 47 CRF19_cpx reference sequences sampled in the United Kingdom (UK) between 2008 and 2010 (S3A and ). These 55 study sequences, range sampled from 2011 to 2016, conformed a well-defined monophyletic cluster in the ML tree as well , not broken down by adding any of the references from LANL or even those mentioned from the UK . In addition, the Bayesian inference of the phylogeny showed that 42 sequences from patients within this outbreak (76.3%) were comprised in seven subclusters of different size (pp≥0.9) (Table 1). All these subclusters had also correspondence in the ML tree, with one or both of the branch-support measures and thresholds considered . Finally, the most recent common ancestor (MRCA) of this outbreak was dated at 2009  by the Bayesian skyline growth with a lognormal relaxed molecular clock, selected as reporting the lowest AICM value (S2 Table).The Bayesian approach confirmed the presence of a local transmission cluster, with an associated posterior probability (pp) of 0.895 . Thus, at least three separate routes of introduction the CRF19_cpx HIV-1 occurred in our area. This pattern was robustly confirmed with the two phylogenetic reconstructions obtained, ML tree  and Bayesian inference.On the other hand, there were two sequences phylogenetically separated from each other and from the rest. One of them was sampled in 2013 and showed a close phylogenetic relation  to reference sequences from Cuba exclusively. The other one, however, sampled in 2016, did not belong to any subgroup  with a high enough confidence value . Outside these clusters but within the outbreak, there are 2 more patients with this mutation. In addition, we also detected in 8 patients (14.0%) the polymorphic mutation V179I/A, though this possesses little direct effect on NNRTI susceptibility. Unlike G190A mutation clusters, the patients with the V179 polymorphism did not show any clear grouping with each other, as depicted in Fig 3 as well as  in Supplementary material.Regarding antiretroviral drug resistance, we found the G190A mutation associated with different levels of non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance in 29 out of 57 patients with the subtype CRF19_cpx (50.9%), including all the patients from clusters A to D  (94.7%). All were Spanish, except two patients from Argentina and one from France. The average age of the cohort was 35.7 years (27.2–42.0). The initial CD4 count was 387 cells/μL (259–468). Eight patients (14.0%) had <200 cells/μL at diagnosis and 26 (45.6%) presented a late diagnosis . In addition, the average first viral load was 4.9 Log10 copies/mL (4.3–5.5), with a zenith value of 5.0 Log10 copies/mL (4.5–5.5), this latter being lower in patients with the G190A mutation . On the other hand, five cases of AIDS (8.8%) were recorded at diagnosis. Only one death was recorded during the study period, due to acute myocardial infarction in a patient two years after his diagnosis and without having any AIDS event during the follow-up period. Finally, 54 patients were being treated with first-line combination ART at the end of the study period, 92.6% of them with viral suppression. Other demographic and clinical data about our cohort as well as the detailed comparison between the groups with and without the G190A mutation are depicted in Table 2.Furthermore, as shown in As far as we know, this study reports the largest cohort of HIV-1 CRF19_cpx outside Cuba. Although the spread of this recombinant outside Cuba has been previously described –11, it iet al. highlighted the high fitness score of the CRF19_cpx subtype in the PR region [The centre of this outbreak is Malaga, with no relation to any other CRF19_cpx sequence from treatment-naïve patients outside this area . The preR region . AlthougCRF19_cpx has also been associated with multiple drug resistance mutations in therapy-naïve Cuban patients , 8. In tFinally, we analysed the demographic and clinical variables in the set of CRF19_cpx patients in our study. These characteristics were similar to those found for non-B infected patients in the Spanish AIDS Research Network Cohort (CoRIS) , except In summary, the CRF19_cpx recombinant has been mainly transmitted as an outbreak over a period of 6 years in southern Spain. Local young MSM are the major risk group affected. This subtype is associated with a high prevalence of cross-class primary resistance to NNRTI in our area, with more than half the cases presenting the G190A resistance mutation. Unlike previous studies, the CRF19_cpx recombinant from Malaga seems less pathogenic, with few cases of AIDS and excellent response to first-line ART.S1 FigThe best substitution model was chosen according to the lowest AIC (Akaike Information Criterion) score as the selection criterion.(TIF)Click here for additional data file.S2 FigReference sequences appear with their corresponding accession numbers while each study patient is represented in red by their sample ID. Numbers near each node show posterior probabilities.(PDF)Click here for additional data file.S3 Fig(A) Phylogenetic relationship of our CRF19_cpx sequences with regards to another 254 reference sequences from the same subtype retrieved from LANL. Each patient is represented in red by their sample ID while reference sequences appear with their corresponding accession numbers. (B) Subtree with the clustering of patients within the outbreak, highlighting in dark grey shading the presence of the G190A mutation as applicable. Asterisks indicate the detection of V179I/A. Only bootstrap proportions ≥50% are shown.(TIF)Click here for additional data file.S4 Fig(A) Phylogenetic relationship of our CRF19_cpx sequences with another 254 reference sequences from the same variant retrieved from LANL. Each patient is represented in red by their sample ID while reference sequences appear with their corresponding accession numbers. (B) Subtree with the clustering of patients within the outbreak, highlighting in dark grey shading the presence of the G190A mutation as applicable. Asterisks indicate the detection of V179I/A. Only SH-aLRT values ≥80% are depicted.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TabletMRCA median and range dates (95% HPD) for each one is depicted.(DOCX)Click here for additional data file.


Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs. Integrase strand transfer inhibitor (INSTI)-based regimens are recommended as first-line combination antiretroviral therapy (cART) in several updated treatment guidelines for HIV after the efficacy of the three INSTIs  has been demonstrated in randomized clinical trials1234567With the widespread use of INSTIs, surveillance of the prevalence of INSTI resistance among ART-naive patients is critical in optimizing ART efficacy, given the concerns that INSTI, particularly raltegravir and elvitegravir, have a relatively lower genetic barrier to emergence of resistance mutations when compared with boosted PIs101110 copies/mL , with 33.1% of the patients having PVL > 5 log10 copies/mL. The mean CD4 count was 299 cells/mm3 , with 31.7% having CD4 counts < 200 cells/mm3.From June 2006 to October 2015, a total of 1370 non-duplicated blood specimens were subjected to genotypic analysis of INSTI resistance at the National Taiwan University Hospital (NTUH). These specimens were from ART-naïve patients (N = 948), ART-experienced patients who had never been exposed to INSTIs (N = 359), and patients receiving raltegravir-containing regimens and experiencing virological failure (N = 63). Baseline characteristics of the patients included in this study are shown in P < 0.001), were younger  and were more likely to be MSM  and infected with subtype B virus , and to have higher mean CD4 count .Compared to ART-experienced/INSTI-naive patients, ART-naïve patients had a higher percentage of male gender . While the first case of INSTI-related resistance mutations was identified in a patient included in November 2006, the majority of the cases (n = 9) were identified in patients included in 2013. The prevalence of INSTI-related resistance mutations was <1% before 2012 , which increased to 1.2% (9/766) in 2013, and gradually declined to 0.5% (1/206) in 2014–15.Among the 1,307 INSTI-naive study subjects, 12 (0.9%) were infected with HIV-1 that exhibited major mutations shown to cause a marked reduction of viral susceptibility to raltegravir and elvitegravir . The preThe 12 INSTI-resistant HIV-1 sequences were all of subtype B; 6 of them were from ART-naive and 6 from ART-experienced/INSTI-naive patients. The amino acid mutations/polymorphisms detected in these individuals are listed in The presence of INSTI resistance mutations was also determined in 63 INSTI-experienced patients. Thirty patients (47.6%) harboured INSTI-related major mutations, which included 17 with Q148H/K/R mutations, eight with N155H mutations, and six with Y143C/H/R mutations. The concurrent major/minor INSTI-related mutations in these sequences are summarized in Beside major INSTI mutations, other integrase substitutions with a Stanford HIVdb score ≥ 10 to at least 1 INSTI, and some polymorphisms that have been reported in previous studies or observed in this study were also determined. Their overall prevalence in INSTI-naive patients and INSTI-experienced patients was 5.2% (69/1307) and 38.1% (24/63), respectively. In INSTI-naive patients, the resistance mutations included L74M (n = 28), E92V (1), Q95K (4), T97A (4), E138AK (3), Y143S (1), V151AL (1), N155S (9), E157Q (11), G163K/R (5), S230R (1) and R263K (4) . The preP < 0.001), nucleos(t)ide reverse-transcriptase inhibitor (NRTIs) , and multi-drug resistance (MDR)  among patients with HIV-1 sequences harbouring INSTI mutations were all significantly higher than those without INSTI major mutations (Among the 1307 HIV-1 specimens from INSTI-naive (948 ART-naive and 359 ART-experienced) and 63 raltegravir-experienced patients, the PR and RT genes were successfully amplified and sequenced in 1292 specimens (94.3%). The baseline demographic and clinical characteristics of the patients with PR and RT genes successfully sequenced were similar to those without analyzable RT/PR sequences (data not shown). Among the 6 samples from ARV-naive patients with INSTI major mutations, only one harboured M184V mutation , known tutations .P = 0.02) and INSTI as the third agent . Accordingly, a significantly lower percentage of them used abacavir/lamivudine as the backbone  and PI as the third agent .When the current regimens used by the ART-experienced patients were analyzed, we found that zidovudine/lamivudine, abacavir/lamivudine, and tenofovir disoproxil fumarate (TDF)/emtricitabine or TDF and lamivudine were the major NRTI backbone antiretrovirals that were used in these patients . The thiPhylogenetic analysis was conducted to determine whether there were clusterings observed among these integrase sequences amplified from INSTI-resistant sequences . Three tIn this study conducted in Taiwan, where raltegravir was only available for clinical use since 2009, we demonstrate that the prevalence of major mutations to INSTI remains relatively low in INSTI-naive patients, 0.6% (6/948) among ART-naive and 1.7% (6/359) among ART-experienced/INSTI-naive patients, which is somewhat different from the absence of major mutations in ART-naive patients reported in the US and Europe1011et al.In patients experiencing virological failure to the first generation of INSTIs, raltegravir and elvitegravir, three genotypic mutation pathways have been defined: Q148H/R/K (±G140S or E138A/K), N155H (±E92Q), and Y143C/H/R (±T97A). Except E92Q, the amino acid substitutions in the parenthesis representing the compensatory mutations that can help restore fully or partly viral fitness1415in vitro studies19et al. in the UKP = 0.02). Whether decreased tolerance to zidovudine/lamivudine may contribute to the risk of accumulating secondary mutations to raltegravir remains to be investigated. Besides the Q148 mutation combined with one or more of G140A/C/S, L74I and E138A/K/T was identified to reduce viral susceptibility to dolutegravir, two amino acid mutations, G118R and R263K, have been reported to confer low-level resistance to dolutegravir22The susceptibility of these INSTI-resistant sequences to the second generation of INSTI, dolutegravir, was also evaluated based on the results from et al. who analyzed 56 specimens from the 278 specimens by 454 sequencing to detect the presence of scarce INSTI-related mutations found that the prevalence of mutations with Stanford HIVdb score ≧ 10 increased to 14.3%Although raltegravir and elvitegravir have a relatively lower genetic barrier than PIs and most NRTIs, the prevalence of primary INSTI resistance mutations is relatively rare based on a few published reports10111011in vitro passage experiment in the presence of raltegravir, and it has never been reported in sequences from patients receiving raltegravirIn INSTI-naïve patients, we observed that 0.7% of the sequences (N = 9) harboured the N155S/T mutation, which was not observed in any of the raltegravir-experienced patients in our study. N155S was first identified by Entamoeba histolytica have been observed in our MSM population in Taiwan25262728In this study, an increased prevalence of INSTI-related genetic mutations was noted in 2013. It could be related to transmission of resistant strains among patients with risky behaviors since increasing incidence of HDV, HCV, syphilis, and The findings of this study should be interpreted with the necessary caution. First, the study was based on patients included mainly in hospitals in northern Taiwan, where the majority of the patients were MSM. While MSM have become the leading risk behavior for HIV infections and TDR is more prevalent among MSM in Taiwan30In conclusion, the prevalence of primary INSTI resistance of the HIV-1 strains identified from ARV-naive patients in Taiwan remains relatively low, which is comparable to the previous reports in high-income countries. Nevertheless, given our previous observation that TDR to other antiretrovirals remains high among MSM in Taiwan, close and vigilant monitoring of INSTI resistance is important before its widespread use in Taiwan.In Taiwan, cART has been provided free of charge since its introduction in April 1997, but routine drug-resistance testing has not been available to clinicians. By the end of December 2015, there were a total of 31,036 people diagnosed as having HIV infection in Taiwan, and about 70% of those who survived were receiving cART. The prevalence of transmitted drug resistance (TDR) in Taiwan has increased from 1999 to 2006, with an overall prevalence of 9.4%TM Test, version 1.5,  and FACSFlow (Becton Dickinson), respectively. The study was approved by the Research Ethics Committee of the National Taiwan University Hospital, Taipei, Taiwan  and the experiment procedures were carried out in accordance with the approved guidelines. The patients who were linked to HIV care at the hospital gave written informed consent for determination of HIV-1 resistance mutations.HIV-infected Taiwanese patients receive HIV care according to the national treatment guidelines at designated hospitals around Taiwan by the Centers of Disease Control in Taiwan. Residual plasma samples for routine determination of plasma viral load (PVL) were obtained from HIV-1-infected patients seeking HIV care at the NTUH. A standardized case record form was used to collect information on demographics, antiretroviral medications, baseline CD4 lymphocyte count, and PVL. PVL and CD4 count were quantified with the use of Cobas Amplicor HIV-1 Monitorhttp://hivdb.stanford.edu; date last accessed, 10 November 2015), in accordance with the drug resistance mutation list of the International AIDS Society-USA Consensus GuidelinesFor those with confirmed HIV infection and linked to clinical care at NTUH, the genotypic resistance assays were performed retrospectively as described previously30integrase coding regions was PCR-amplified as described previouslypol2950A (5′-TCA KCA CCT GCC ATC TGT TTT CC-3′)/2064A (5′-AYA ARG GRA TTG GAG GAA ATG AAC A-3′). The amplification condition was 35 cycles of 94 °C for 15 s, 55 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 7 min. The major INSTI mutations, which have been determined having a marked reduction of viral susceptibility to raltegravir and elvitegravir, include Y143C/H/R, Q148H/K/R, and N155H. Besides these three major mutations, the integrase substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were included, such as H51Y, T66A/I/K, L74M, E92G/Q/V, Q95K, T97A, F121Y, E138A/K, G140S/C/A, Y143G/K/S/A, P145S, Q146P, S147G, V151A/L, S153F/Y, N155S/T, E157Q, G163 K/R, S230R, and R263K. Some amino acid substitutions  that have been described in more than 2% of the study population in previous studies1113To determine the resistance mutations related to INSTIs, an 884-bp fragment of http://hiv-web.lanl.gov/seq-db.html). Sequences were aligned with the Clustal W listed in the MEGA  analytical package (version 3.0)Reference sequences of various subtypes and recombinants were retrieved from the Los Alamos database . Categorical data were analyzed using chi-square or Fisher’s exact tests, as appropriate, and continuous variables were compared using the Wilcoxon test. All tests were two-tailed and a P value < 0.05 was considered significant.How to cite this article: Chang, S.-Y. et al. Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan. Sci. Rep. 6, 35779; doi: 10.1038/srep35779 (2016).


Polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC), monocytic myeloid-derived suppressor cell (M-MDSC), and the Breg frequencies were comparable to those of patients with a 3-drug ART. However, the Treg levels were significantly lower in the NE patients than those in 3ART-treated individuals and other control groups (p ≤ 0.0033). The Gag/Nef-specific CD8 T cell response was broader (p = 0.0134) with a higher magnitude (p = 0.026) in the NE population than that in the patients with conventional ART. However, we did not find a correlation between the frequency of the immune suppressive cells and the interferon-gamma+ CD8 T cell response. In the treatment intensification subjects, the frequencies of the immune suppressive cells were comparable or lower than those of the conventional ART-treated subjects, with surprisingly broad HIV-specific CD8 T cell responses, suggesting a preservation of immune function with the applied treatment regimen. Interestingly, these effects were seen in both treatment intensification subpopulations and were not attributed to the start of treatment in primary infection.In untreated HIV infection, the efficacy of T cell responses decreases over the disease course, resulting in disease progression. The reasons for this development are not completely understood. However, immunosuppressive cells are supposedly crucially involved. Treatment strategies to avoid the induction of these cells preserve immune functions and are therefore the object of intense research efforts. In this study, we assessed the effect of treatment intensification [=5-drug antiretroviral therapy (ART)] on the development of suppressive cell subsets. The New Era (NE) study recruited patients with primary HIV infection (PHI) or chronically HIV-infected patients with conventional ART (CHI) and applied an intensified 5-drug regimen containing maraviroc and raltegravir for several years. We compared the frequencies of the immune suppressive cells, namely, the myeloid-derived suppressor cells (MDSCs), regulatory B cells (Bregs), and regulatory T cells (Tregs), of the treatment intensification patients to the control groups, especially to the patients with conventional 3-drug ART, and analyzed the Gag/Nef-specific CD8 T cell responses. There were no differences between PHI and CHI in the NE population ( Since 2010, the number of HIV-infected people with access to highly active antiretroviral therapy (ART) increased from 7.5 to 18.2 million in 2016. However, with 36 million people living globally with HIV, only half of them are treated despite intense efforts toward access to ART  in plasma, but an eradication of the virus has not been achieved so far. Within the New Era (NE) study, treatment intensification by adding raltegravir and maraviroc to a 3-drug regimen has been investigated with the goal of eradication or at least a functional cure. The study recruited patients with primary HIV infection (PHI) or patients with chronic HIV infection (CHI). PHI was defined by less than two bands in a Western blot analysis. CHI patients were already treated successfully for at least 36 months with a protease inhibitor (PI) based conventional ART. All the patients had a CD4 nadir above 200 cells/μl and no evidence of resistance to the PI-based regimen. The aim of the study was to stop residual viremia, as well as to reduce and limit the viral reservoir measured by proviral DNA , 6. HoweIn early untreated HIV infection, a strong cytotoxic CD8 T lymphocyte response (CTL)—among others—effectively decreases VL –11. HoweIn HIV infection, the role of Tregs is ambiguous and has not been evaluated conclusively –25. On tMDSCs are immature cells with a myeloid origin, which act immunosuppressively by inhibiting the functions of T lymphocytes, among others. MDSCs are divided into two major subsets, including polymorphonuclear (PMN) and monocytic (M) MDSCs , 27. In + Breg frequencies are significantly elevated in viremic patients compared with HIV-uninfected individuals  and the German federal institute for drugs and medical devices (BfARM)] and 92 individuals within 4 control groups . The treatment intensification within the NE study consisted of two nucleoside reverse transcriptase inhibitors, a PI, and maraviroc and raltegravir in all cases. Patients with a virus with CXCR4 tropism were excluded from the study.n = 10).Primary HIV infection .Chronic HIV infection  of all the NE patients was 5.5 years. Thus, CHI had a median ART duration of 11/9.5–16.5 years (median/range).n = 27): CD4 cell count below 400/μl, VL above 10,000 copies/ml.Progressors (PR) : CD4 cell count above 500/μl and VL below 2,000 copies/ml .Controllers (CO) : any CD4 cell count and VL below 50 copies/ml. The median duration of treatment: 10.5/4–18.5 years (median/range). One individual in this group was treated with maraviroc, but no one was treated with raltegravir. No threshold was set for the CD4 count, because this was not done in any of the treated groups.3ART .HIV-uninfected controls (HC)  with a VL below 40 copies/ml and a CD4 count of 467 cells/μl in the absence of ART.None of the ART-treated patients experienced treatment failure.All the patients on any treatment regimen were summarized in the subgroup with ART (w ART) and in analogous patients untreated in the subgroup without ART (w/o ART).To further interpret our data, we stratified the cohort in the following subgroups:The clinical characteristics of the study subjects are shown in Table Peripheral blood mononuclear cells were isolated from freshly obtained EDTA blood by Ficoll density gradient centrifugation . The frequencies of the PMN-MDSCs and M-MDSCs (including isotype control) were determined in fresh PBMCs directly after blood draw (≤2 h after blood draw). For this reason, only individuals living in Munich were included in the MDSC studies. The frequencies of the Bregs and Tregs as well as the IL-10 production by the Bregs and the T cell immune responses by interferon-gamma enzyme-linked immunospot (Elispot) assay were determined using frozen PBMCs from all the study subjects.+, CD24hi, and CD38hi  or frozen (Tregs/Bregs) PBMCs as previously described . The fold CD38hi , 33, and6 PBMCs/ml) were incubated for 48 h with 10 µg/ml CPG-B (oligodeoxynucleotide-2006), 2 µg/ml PAM , and 2 µg/ml CD40L  at 37°C and in 5.0% CO2, and during the last 5 h, the cells were supplemented with 1 µg/ml ionomycin, 50 ng/ml phorbol-12-myristate-13-acetate (PMA), 50 µg/ml brefeldin A , and 1 µl/ml monensin (BD Golgi Stop™ BD Biosciences). Second, we only stimulated the cells with ionomycin/PMA. After 2 h of incubation at 37°C/5.0% CO2, brefeldin A and monensin were added, and the cells were incubated for another 5 h. After the incubation, the cells were washed and stained intracellularly for IL-10. The background range was <0.02% for IL-10 production.The Bregs show an immunosuppressive function by the production of IL-10. For measuring IL-10 production, we stimulated the thawed PBMCs of five PR and five HC in two ways . FiOverlapping synthetic peptides corresponding to the HIV proteins Gag and Nef were used for screening . The peptides had a purity of ≥70%.5/well) and peptides  as described previously , we also did pairwise Mann–Whitney U tests with Bonferroni Correction for multiple testing. Four pairwise comparisons were considered relevant and were tested for each experiment as follows: NE vs. 3ART; NE vs. HC; NE vs. PR; and NE vs. CO. The corrected level of significance, therefore, was p < 0.0125 when HC was included, and it was p < 0.0167 in the experiments without HC. Spearman rank test was used for correlation analyses and Wilcoxon signed rank test for paired comparisons (level of significance p < 0.05). Only tests with significant results are indicated in the figures.The statistical analyses were performed using GraphPad Prism version 5.0. In most cases, our data were not normally distributed. Therefore, we only used non-parametric tests. Comparisons between two groups were done with the Mann–Whitney p = 0.002). However, there was no difference to the 3ART group (p = 0.65)    Figure B. We furThus, our analysis shows low PMN-MDSC frequencies in the treatment intensification-treated individuals, which were, however, comparable to the 3ART patients. In addition, all the ART-treated subjects had PMN-MDSC levels that did not reach the level of the HIV-uninfected controls.p = 0.008)  (data not shown). In contrast to the PMN-MDSCs, the percentages of M-MDSCs in our cohort were significantly higher in the treatment intensification patients than those in the HC patients (p < 0.0001) but were comparable to those in the 3ART patients (p = 0.21)    Figure A, whereats p = 0.1  agonists p = 0.002; Figure S2 in Supplementary Material]. Overall these results showed that, in our assays, phenotypically defined Bregs are capable of IL-10 production, and therefore, they are Bregs. As previously described, we observed a positive correlation of Breg frequencies with VL in viremic HIV-infected patients    , and these frequencies were comparable to those of the HC group (p = 0.61)  , whereas the difference compared with the HC group failed to reach statistical significance    (data not shown). A higher median CD4 cell count in the PHI subgroup than that in CHI  along with the abovementioned comparable Treg frequencies resulted in higher absolute Treg cell counts in the PHI groups compared with those in the CHI group, which just missed statistical significance (p = 0.08)  .Immune suppressive cells inhibit CD8 T cell responses. Therefore, we evaluated if there was a correlation between the HIV-specific CD8 T cell responses and the levels of immune suppressive cells studied here for the patients treated with a 5-drug ART. However, we were not able to demonstrate any correlation between the breadth or magnitude of interferon-gammaA direct association between the levels of inhibitory immune cells and the interferon-gamma positive CD8 T cell response was not found in the treatment intensification.p = 0.0134)  . This suggested that it was a very special group that was selected for the CHI group in the NE study.Interestingly, the NE patients revealed a higher number of CD8 T cell responses in Gag and Nef than that of the 3ART patients  Figure A. In con) Figure B, showin6 PBMCs (median 700) and was therefore significantly higher compared with that of the 3ART patients (p = 0.026)   Figure C. In con) Figure D. Thus, Looking at the distribution of the CD8 T cell responses within the viral proteins, Gag p17 and Gag p15 were more often recognized by the CO group and, interestingly, also by the NE group than by the other groups. Studying this finding in detail showed that the CHI group recognized the Gag subunits p17 and p15, and the PHI group did not. Therefore, the CHI patients were similar to the CO patients in this respect. Gag p24 and Nef were the most frequently recognized protein subunit within all the groups as was to be expected  and was not attributed to an early start of ART in primary infection. In addition, the NE population showed broader and stronger HIV-specific CD8 T cell responses, mainly driven by the CHI subgroup.Myeloid-derived suppressor cell levels are significantly elevated in chronic progressive HIV infection. This association is shown for both subsets of MDSCs, namely, PMN-MDSCs , 41 and The results for the M-MDSCs are not as straightforward. In our study, there was no difference for these cells in any of the HIV-infected groups (including PR), and all the HIV-infected patients had higher M-MDSC levels than those of the HC group. This is in contrast to data published by others , 42. One+CD24hiCD38hi)  , 33. HowiCD38hi) . In chroiCD38hi) , 32, 43.iCD38hi) , 32. Ourp = 0.34) and were significantly lower than those in PR and 3ART patients (p < 0.009). Surprisingly, the lowest relative Treg frequencies were found in the NE patients with comparable levels in PHI and CHI. The difference between the NE patients and 3ART patients was especially remarkable (p < 0.0001), as the levels for those two groups were rather similar for all other cell types. Therefore, treatment intensification seems to have a beneficial effect for this specific cell type. Another reason for this finding could be the inclusion of maraviroc to intensify the treatment in the NE patients. Two studies analyzed the influence of maraviroc on Treg frequencies with contrary results . However, this finding, as it stands, questions the necessity for treatment start in acute HIV infection with regard to immune suppressive cells. On the other hand, the CHI patients in the NE study were chosen very selectively. The patients had to be virologically suppressed by a PI-based ART for at least 3 years and without a history of virological failure or blip , 3. It iIn our study, we evaluated the CD8 T cell responses on an epitope level to assess possible correlations between the suppressive cells and the breadth of these responses. A limiting factor for this analysis is that the NE patients were treated for several years, and it is known that CD8 T cell responses decline both in breadth and magnitude after the start of ART with the disappearance of viremia . We restGag p17 and p15 contain more variable regions and, therefore, are often less recognized in HIV-infected individuals , 49, 53.With this study, we comprehensively described several immunosuppressive cell subsets in a unique patient population. However, there were some limitations. First, the correlations might not have reached significance because of the small group sizes. The NE study was a multi-center study with a necessity for overnight shipment of samples for some centers. Because of the fragile nature of MDSCs –56, we wAmong the investigators of the study, it was highly discussed whether to stop treatment in this study population or not. In addition, if the treatment was stopped, should this be done with an additional intervention  or without. We think that it would be particularly interesting to stop treatment in the CHI group because, according to our results, the patients of this group seem very special as described earlier.This study was carried out in accordance with the recommendations of the Institutional Review Board of the Ludwig-Maximilians-Universität, Munich, Germany with written informed consent from all subjects. Samples of New Era patients were obtained within the scope of an amendment of the New Era study  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the Ludwig-Maximilians-Universität, Munich, Germany.EMG designed the research, acquired the data, performed the experiments, analyzed the data, performed the statistical analysis, and drafted the manuscript. TH acquired the data, performed the experiments, analyzed the data, and performed the statistical analysis. RS and AN acquired the data and performed the experiments. RP, AU, and JB provided samples and edited the manuscript. EW, EG, and HJ provided samples, contributed to the interpretation of the results, and edited the manuscript. JRB provided samples, interpreted the data, and wrote and edited the manuscript. RD designed and oversaw the research, analyzed and interpreted the data, performed the statistical analysis, wrote the manuscript, and revised the final version of the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.