Simplified maintenance therapy with ritonavir-boosted atazanavir (ATV/r) provides an alternative treatment option for HIV-1 infection that spares nucleoside analogs (NRTI) for future use and decreased toxicity. We hypothesized that the level of immune activation (IA) and recovery of lymphocyte populations could influence virologic outcomes after regimen simplification.+CD56+CD16+) cells and cell-associated markers .Thirty-four participants with virologic suppression ≥48 weeks on antiretroviral therapy (2 NRTI plus protease inhibitor) were switched to ATV/r alone in the context of the ACTG 5201 clinical trial. Flow cytometric analyses were performed on PBMC isolated from 25 patients with available samples, of which 24 had lymphocyte recovery sufficient for this study. Assessments included enumeration of T-cells (CD4/CD8), natural killer (NK)  >50 copies/mL during the study. NK cell levels below the group median of 7.1% at study entry were associated with development of VL >50 copies/mL following simplification by regression and survival analyses (p = 0.043 and 0.023), with an odds ratio of 10.3 (95% CI: 1.92–55.3). Simplification was associated with transient increases in naïve and CD25Lower NK cell levels prior to regimen simplification were predictive of virologic rebound after discontinuation of nucleoside analogs. Regimen simplification did not have a sustained impact on markers of IA or T lymphocyte populations in 48 weeks of clinical monitoring.NCT00084019ClinicalTrials.gov  Trials of antiretroviral treatment (ART) simplification to ritonavir-boosted protease inhibitors (PI) alone have shown mixed success, with some trials mirroring the outcome of standard triple therapy and others failing to show equivalence.+ T-cell counts have increased following antiretroviral therapy and levels of HIV-1 RNA have been suppressed for a predetermined time period. The A5201 study used an entry criterion of ≥250 CD4+ T cells/mm3 to minimize risk of insufficient immune recovery prior to maintenance treatment simplification, similar to other maintenance simplification studies.+ T cell counts and suboptimal medication adherence.+ T cell counts were also associated with virologic failure, but in patients that had only been suppressed for 3 months prior to treatment simplification to LPV/r alone.We assessed the immunologic determinants of sustained virologic suppression in the AIDS Clinical Trials Group (ACTG) protocol A5201. This was a prospective, open-label, single-arm pilot trial of simplified maintenance therapy with atazanavir-ritonavir (ATV/r) alone after prolonged virologic suppression.Our study therefore assessed the immune profile of patients before regimen simplification, the impact of regimen simplification on the T and NK cell populations and immune activation, and whether these immunologic parameters correlated with levels of residual viremia, measured by single copy assay, and detectable viremia above 50 copies/mL.6 peripheral blood mononuclear cells (PBMC) were obtained and stored at −140°C. Of the 25 patients with available PBMC samples, one was excluded from the analyses due to poor sample quality (<10% viable PBMC from expected). Most of the PBMC samples were collected at the same timepoints as the HIV-1 viral load samples. All virologic failures as defined in the A5201 clinical trial had available PBMCs and underwent flow cytometry analyses. Clinical trial data including virologic outcomes, CD4 nadir and HIV-1 RNA levels were included in our analyses to assess for potential impact on outcomes or in immune parameters. In all participants, HIV-1 RNA levels were measured with the Roche Amplicor Cobas v1.5 assay. To assess the impact of treatment simplification on HIV-1 residual viremia in patients with long standing HIV-1 suppression, the single copy assay (SCA), having a limit of detection of 1.1 copies/mL as reported by Palmer et al, was used in a subset of 13 participants, which included all 4 participants with study defined virologic failure and 9 participants who had been previously amplified efficiently by SCA in a prior study.The Institutional Review Boards of all the participating and enrolling institutions listed in the acknowledgements approved the A5201 study and each participant provided written informed consent, these include: the University of Colorado Health Sciences Center, Duke University, Stanford University, the University of Nebraska Medical Center, Weill-Cornell Medical College, the University of Pittsburgh, the University of Cincinnati, the University of Hawaii–Manoa, the University of Iowa, the University of North Carolina–Chapel Hill, the University of Texas–Southwestern Medical Center, and the University of Puerto Rico. Thirty-four participants underwent regimen simplification to ATV/r during the A5201 study. Participants included in the study were receiving a protease inhibitor plus at least 2 NRTIs with plasma HIV-1 RNA suppression below 50 copies/mL for at least 48 weeks immediately prior to study entry.Prior studies of PI monotherapy/simplification using lopinavir/ritonavir have shown an increased prevalence of detectable HIV-1 RNA above 50 copies/mL; persistently detectable HIV-1 RNA levels may potentially lead to virologic failure and emergence of HIV resistance.6 cells were incubated in FACS buffer containing 2%BSA and 0.1% NaN3 along with appropriate combinations of antibodies for 30 minutes following manufacturer's recommendations. The cells were then washed and resuspended in FACS buffer. Flow cytometry data was immediately acquired by using the LSR II flow cytometer (BD Biosciences), in accordance to the manufacturer's instructions. The antibodies used were purchased from BD Biosciences , Beckman-Coulter (anti-CD8-ECD), eBioscience (anti-CCR7-APC-AF700) and BioLegend (anti-CD56-AF700). Additional negative controls were included for each sample during staining and acquisition steps. The parent cells were the CD3+ T cell lymphocytes for our analyses and all other T and NK cell percentages are based from this parent population. A gate was set on the population of singlet events as determined by the linear relationship between forward scatter height and area. This was followed by a gate on the live population of lymphocytes based on forward and side scatter light properties. The CD56 and CD3 parameters were plotted, and a gate was set on the NK cells based on their classical definition of being CD56+/CD3- lymphocytes. All flow cytometry gating and data were reviewed by both flow cytometry technicians (two) and by the principal investigator to confirm the validity of the samples, the gating strategy and the final numbers, prior to inclusion into the final data set.PBMC samples collected as part of the A5201 protocol at entry, weeks 6, 18, 30 and 54 were used for the analyses. We expanded the flow cytometry testing to include both T cell and NK cell populations, as the latter is associated with early viremic control and may contribute to the control of HIV-1 viremia in patients on ART. The impact of the NK cell population on HIV-1 residual viremia has not been previously evaluated. Cryopreserved PBMC samples were thawed and cell viability was determined using a Vi-CELL analyzer  with a target of ≥75% viability for each sample to be used for testing. For each timepoint, 1.5×10All statistical analyses were performed using SPSS statistics 20 (IBM). Sample size was limited to patients participating in the A5201 single arm study with available PBMC samples for analyses. We used both parametric analyses including means test, t-test, and non-parametric analyses including median test, the Mann-Whitney-U, mixed model analyses, and Cox multivariate regression analysis and cumulative survival analyses. Both parametric and non-parametric test were used as appropriate. Univariate analyses were used to examine potential predictors of sustained virologic suppression with p-values ≤ 0.1 which were subsequently analyzed using both forward and backward multivariate regression analysis. Multivariate analyses was used to determine if any of the factors were true predictors of virologic outcome and to eliminate any potential bias or confounding of multiple comparisons/repeated testing producing a false positive finding.+ T cell count of 616 (range: 443–756) cells/mm3. We compared the baseline T and NK cell parameters between participants maintaining HIV-1 RNA suppression and those who did not (HIV-1 RNA > 50 copies/mL). At baseline, the CD4+ and CD8+ T cell populations were similar for naïve, central memory, effector memory and CD25+ T cells between participants who maintained suppression of HIV-1 RNA and those with detectable HIV-1 RNA , and for co-expression of CD56+CD16+ with mean value of 11.5% versus 3.2% (p<0.01). T cells expressing NK cell associated marker CD56+ cell levels were also higher in participants with sustained viral load (VL) suppression as compared to participants with detectable VL, with median values of 7.2% versus 4.3% (p<0.01) (Participants entering the study had a median CD4(p<0.01) . NK cell(p<0.01) . Higher +CD38+HLADR+, CD8+CD69+, and CD3−CD56+CD69+ percentages were higher in the participants whose HIV-1 RNA levels remained suppressed below 50 copies/mL during the study. In comparison of participants with undetectable versus detectable viremia, the median percentages for CD4+HLADR were 13.6% vs. 4.2% (p = 0.01), CD4+CD69+ were 12.2% versus 6.3% (p = 0.02), CD4+CD38+HLADR+ were 3.6% versus 1.20% (p = 0.07), CD8+HLADR+ were 16.8% versus 6.1% (p<0.01), CD8+CD69+ were 8.4% versus 3.7% (p = 0.03) and for CD56+CD69+ were 27.0% versus 15.9% (p = 0.04) (Significant differences in T cell immune activation at baseline were seen between the two groups. CD4 = 0.04) . The low+CD69+, CD4+CD38+HLA-DR+, CD8+HLA-DR+, CD8+CD69+, CD8+CD279+, CD3+CD56+, CD3−CD56+, CD3−CD56+CD16+, CD3−CD56+CD69+ and CD3−CD56+CD158a+. These immunologic variables were included in a multivariable regression analysis with forward and backward regression. We also included participant demographic characteristics, including age and race, and nadir CD4+ T cell count prior to start of antiretroviral treatment. Only one parameter-population remained in the model, CD3−CD56+CD16+, with a p-value of 0.043 in forward regression and 0.034 in backwards regression analyses. NK cells expressing the CD69+ cell-associated marker of early activation did not remain in the model but the p-value neared statistical significance at 0.055  but the difference was not present by week 54 (p = 0.67).Cox univariate regression analyses of baseline immunologic parameters as continuous variables were performed to assess for any associations with the development of detectable HIV-1 RNA levels above 50 copies/mL. Only 9 of the T or NK cell populations or immune activation parameters analyzed had a p-value of ≤0.1 in univariate regression, including CD4at 0.055 . Cumulatat 0.055 . The odd+ and CD94+ markers . Other parameters were not significantly correlated (data not shown).Levels of T and NK cell populations and immune activation markers were analyzed with HIV-1 RNA levels using both the Single Copy Assay (SCA) and/or the Roche Amplicor v1.5 assay. During the trial, blood samples with detectable HIV-1 RNA levels below 50 copies/mL by SCA correlated with higher NK cells expressing CD158b+CD45RO+CCR7+CD27− , CD8+CD38+HLA-DR+ , NK cells CD3−CD56+CD16+  and CD56+CD94+ , in univariate analyses , but by study week 54 the levels were similar to baseline (p = ns). The CD4+CD45RO+CCR7+CD27− cell population also showed a trend to increase following simplification in the participants with suppressed viremia during the study but did not reach statistical significance (p = 0.073). No statistically significant difference was seen in immune activation levels following treatment simplification in either outcome group. All other T and NK cell markers were not statistically associated with treatment simplification .Comparisons of the T and NK cell populations and levels of IA markers before and after treatment simplification revealed no impact of treatment simplification on the immune parameters examined. Participants with suppressed viremia during the study tended to have an increase in the percentage of CD4We examined the impact of baseline T and NK cell populations and markers of immune activation on virologic outcomes following regimen simplification to atazanavir/ritonavir alone. In addition, the impact of regimen simplification on immunologic parameters was investigated. Our study is the first to measure a possible predictive value of baseline NK cell levels for virologic outcome. Participants with NK cell levels below the median had 10 times higher risk of developing HIV-1 RNA levels above 50 copies/mL during the study. The difference in NK cell levels was seen in our study group while patients were still on their original combination antiretroviral therapy and before any intervention had occurred. Increased incidence of detectable viremia above 50 copies/mL has been reported in other trials of treatment simplification strategies and has led to a concern for increased risk of virologic failure and possible development of drug resistance mutations. + T cell levels prior to study entry does not provide a complete assessment of functional immune status reconstitution, and duration of HIV-1 RNA suppression may not be sufficient as an entry criterion for trials of maintenance therapy. Other parameters that were associated with detectable HIV-1 viremia at some point during the study included markers of immune activation HLA-DR and CD38, which could be indicators of undetected viral replication and/or ongoing inflammation, and in turn impact treatment outcomes. These markers reached statistical significance in univariate analyses but did not remain in the model as significant factors in multivariate analyses, possibly due to the limited sample size of our study. Larger studies are needed to further evaluate if the cell markers not retained in the regression models were affected by the limited sample size and have a significant impact on ART treatment responses.The NK cell levels and immune activation marker differences at baseline demonstrate that despite prolonged HIV-1 RNA suppression on antiretroviral therapy, participants had significant differences in immunologic parameters at entry into the study. Therefore the immune status of patients entering HIV clinical trials as currently assessed by CD4+ naïve and CD4+CD25+ T cells in participants with virologic suppression during the trial but the differences were not sustained after 48 weeks of simplification. In the OK04 trial regimen simplification to LPV/r was associated with a modest increase in CD4+ T cells at 48 weeks.+ T cells increases were not detected in our study as previously reported.Regimen simplification to atazanavir/ritonavir was not found to be an independent predictor of changes in either T or NK cell populations or on the levels of immune activation markers. We observed initial gains in CD4+ and CD94+ surface cell markers tended to increase with rising residual viremia by SCA. Once the HIV-1 RNA levels were between 50–99 copies/mL, then CD4+ central memory T cells, NK cells and markers of inflammation tended to decrease as compared to samples from participants with suppressed HIV-1 RNA levels. These paradoxical decreases in markers of inflammation could be related to migration of activated cells from the circulation to the periphery as HIV-1 viral replication expands. These findings may be relevant as we monitor patients with low level detectable viremia or viral blips, as these could signify that the low level increase in plasma HIV-1 RNA is due to actual replication instead of assay variability.HIV-1 residual viremia below 50 copies/mL and low level viremia between 50–100 copies/mL were associated with distinct changes in immune cell populations and activation markers. NK cell CD158b+ T and NK cell responses.+ and CD8+ T cell responses to Gag peptides and NK cell responses to Env and Reg peptides have been correlated with improved viremia control and higher CD4+ T cell counts.+ and CD8+ T cell responses.bright subsets, NKp30 and NKp46 expression in NK cells have been associated with reduced activation and cytotoxic activity with progressive HIV-1 infection.+ T cell recovery as it is on regimen potency.Both the innate and adaptive immune systems have a role in the host response against HIV-1 viral replication. Initial viremia control in acute infection is achieved by cytotoxic activity of CD8The A5201 study was designed as a single arm study, which limits our ability to compare our results to control participants not undergoing treatment simplification. SCA HIV-1 viral load data are also limited to 13 participants who were known to have amplifiable HIV-1 gag sequences, limiting the depth of the analyses below 50 copies/mL. Low level detectable HIV-1 viremia above 50 copies/mL seen in this study may represent either new full rounds of replications due to incomplete suppression or viral shedding from previously infected cells. In the A5201 study, no PI resistance mutations were detected by either standard or single genome sequencing to support possible viral evolution and therefore new rounds of replication. Poor medication adherence will impact virologic rebound and was documented in at least two A5201 study participants. However, most study participants indicated good medication adherence during the trial and had been suppressed for longer to 12 months prior to study entry.+ T cell counts on average above 600 cells/mm3, and even after adjusting for nadir CD4+ T cell counts. Different levels of immune reconstitution and activation may explain the differences in treatment outcomes in trials of maintenance regimen simplification, and could explain why some patients may not require triple combination therapy to maintain HIV-1 suppression. Future trials of maintenance therapy strategies should include more in-depth immune assessments to further confirm these findings and better understand the relationship between the NK cell populations and other immune markers and treatment outcomes. Baseline assessment of NK cell levels and immune activation markers could provide information for early detection of patients at increased risk of virologic failure.In summary, this study demonstrates that patients on suppressive ART for similar timeframes have distinct immune cell populations and levels of cellular activation, despite CD4


There are no data comparing the response to PI/r-based regimens in people presenting for care with low CD4 counts or AIDS (LC).To compare the response to LPV/r-, DRV/r- or ATV/r-based cART regimens in LC initiating cART from ART-naive.3 (low CD4-LC) or CD4 counts ≤200 cells/mm3 (very low CD4-VLC) and/or AIDS, starting their first PI/r-based regimen after 2008. Initial regimens were compared by intention-to-treat: i) time to viral failure (VF) (first of 2 consecutive VL>200 copies/mL after≥6 months); II) time to PI/r discontinuation/switching for any cause (TD) and for toxicity (TDT); III) treatment failure (TF) (VF or TD). Kaplan-Meier and Cox analyses were used.We included people enrolled in Icona with either CD4 counts ≤350 cells/mm1,362 LC patients were included ; 813 VLC. In a median of 18 months (IQR:7–35), the 1-year probability of VF and TF were 2.8% (1.9–3.8) and 21.1% (18.7–23.4). In the adjusted analysis, patients initiating ATV/r had a 53% lower chance, and those initiating DRV/r a 61% lower chance of TD, as compared to LPV/r; the risk of TF was more likely in people starting LPV/r. Results were similar among VLC; in this subgroup LPV/r including regimens demonstrated a lower chance of VF.We confirmed in LC a low chance of virological failure by 1 year, with small differences according to PI/r. However, larger differences were observed when comparing longer-term endpoints such as treatment failure. These results are important for people presenting late for care. The recently published START trial has demonstrated that antiretroviral therapy (cART) should be started as early as possible after HIV diagnosis ; this is3 or individuals with a even more advanced stage of HIV disease (a diagnosis of AIDS and/or CD4 counts of less than 200 cells/mm3) at the time of their first presentation for ´. Details of the study are described elsewhere [ICONA Foundation Study (ICONA) is a multi-centre prospective observational study of HIV-1-infected patients, which was set up in 1997. Eligible patients are those starting cART when they are naive to antiretrovirals, regardless of the reason for which they had never been previously treated and of the stage of their disease. The ICONA Foundation study has been approved by IRB of all the participating centers; sensitive data from patients are seen only in aggregate form. All patients sign a consent form to participate in ICONA, in accordance with the ethical standards of the committee on human experimentation and the Helsinki Declaration (1983 revision). Demographic, clinical and laboratory data and information on therapy are collected for all participants and recorded using electronic data collection [lsewhere .We focussed on two groups of individuals using the historical definitions of late presenters as developed by an International Consensus:3 (low CD4 count group -LC)diagnosis of AIDS and/or CD4 count ≤350 cells/mm3 (very low CD4 count -VLC)diagnosis of AIDS and/or CD4 count ≤200 cells/mmWithin these groups, we selected individuals who initiated their initial cART regimen when there were still ART-naïve after 31/12/2008. These initial cART had to include two nuclesoside reverse transcriptase inhibitors  or abacavir+lamivudine (ABC/3TC) + either ritonavir-boosted lopinavir (LPV/r), or ritonavir-boosted atazanavir (ATV/r), or ritonavir-boosted darunavir (DRV/r). In addition, individuals had to have at least one month of clinical follow-up to be included in the analysis.The response to these initial regimens was compared according to the specific PI/r started with respect of four main outcomes:time to virological failure (VF) defined at time of the first of 2 consecutive VL>200 copies/mL after ≥6 months of ART;time to treatment discontinuation/switching of the PI/r component of the regimen regardless of the reason (TD) anddiscontinuation because of toxicity (TDT);treatment failure (TF) defined as time to VF or to discontinuation/switching of the PI/r component of the regimen.All causes of discontinuation are collected in the ICONA database as reported by the treating physician. In particular, clinicians are asked to report for each single drug which was the main reason for discontinuing it. These reasons include the broad categories of simplification (defined either as the reduction of drugs included in the regimen or the decrease in daily doses or pills), intolerance, toxicity, failure , non-adherence, planned interruption  and other causes .The main exposure of interest was the specific PI/r initiated and a separate Cox regression model was fitted for the risk of VF, TD, TDT, and TF. The same set of potential confounders were considered for all four models which included: age, gender, comorbidities, calendar year of starting cART, nadir lymphocyte T CD4+ cell count, HIV-RNA plasma level at cART initiation, whether TDF/FTC or ABC/3TC were used in the initial regimen.The main characteristics of the two study populations (LC and VLC) have been described and compared according to the initial PI/r started. Prevalence in the categorical variables has been compared across the strata using a chi-square test. Continuous variables have been compared using non-parametric tests.The response to initial regimen was compared using an intention-to-treat analysis with respect to the four main outcomes described in the previous paragraph .Standard survival analysis by Kaplan-Meier (KM) curves and Cox regression models was used; KM estimates with 95% confidence intervals at 1 and 3 years from cART initiation were computed and KM curves shown; unadjusted and adjusted relative hazards (RH) were computed and reported. Confounding was addressed by standard multivariable modelling including potential confounders in the adjusted models. Cox regression models were further stratified for clinical site of enrolment. All analyses have been performed using SAS version 9.4.A total of 1,362 LC patients fulfilling the inclusion criteria were analysed: 607 (44.6%) received DRV/r, 552 (40.5%) ATV/r and 203 (14.9%) LPV/r-including regimens. Characteristics of LC patients according to PI-including regimen are described on DRV/r was given mainly as QD regimen (in 489 patients-80.6%), but 87 patients (14.3%) were given 600mg BID dosage and in 31 (5.1%) the scheduled dosage was unknown.A total of 813 of the 1,362 patients (59.7%) fulfilled the definition of presenters with very low CD4 count (VLC); 414 (50.9%) initiated DRV/r-, 268  initiated ATV/r- and 131 (16.1%) initiated LPV/r-including regimens. Differences among the treatment groups are shown in Over a median follow-up of 18 months (IQR:7–35), 57 LC patients (4.2%) experienced virological failure (VF), 507 (37.2%) experienced treatment discontinuation (TD), 97 (7.1%) discontinuation because of toxicity (TDT) and 485 (35.6%) treatment failure (TF).The Kaplan Meier (KM) curves of reaching each of the end-points are represented in Looking in detail at the different end-points, the KM estimate of VF was 2.8% (95%CI: 1.9–3.8) at 1 year and 5.0% (95%CI: 3.6–6.4) at 3 years; there were no differences in the probability of VF according to the PI/r of the regimen  there weIn the multivariable Cox model , taking When only the 813 VLC patients were included, in a median follow-up of 15 (6–33) months, 28 patients underwent VF, 162 discontinued the PI/r of their regimen, 28 discontinued because of toxicity and TF occurred in 167 patients. The 1 year-probability of these events was 4.3 (2.7–5.9)% for VF, 22.4 (19.4–25.5)% for TD, 3.9 (2.5–5.4)% for TDT, and 24.2 (21.0–27.4)% for TF.In these VLC patients, risk of VF was significantly higher for those receiving ATV/r including regimens as compared to LPV/r, in the multivariate model. Similar to the findings among the LC group, both ATV/r and DRV/r showed a lower chance to discontinue the PI component of the regimen as compared to LPV/r including regimens.DRV/r showed a significantly 69% lower chance (p = .081) of discontinuing the PI because of toxicity as compared to LPV/r. Finally, the chance of TF was significantly lower for both ATV/r (by 44%) and DRV/r (by 66%) as compared to LPV/r .3 or AIDS respectively. Further, the probability of discontinuation of the PI/r component of the regimen is of 19.7% by one year, similar to what already published on the Icona cohort [In our cohort including only severely immunodepressed patients given a first line PI/r containing therapy, we were able to demonstrate several differences in risk of virological failure, tolerability and durability of the different regimens. But first of all, also in these categories of severely immunodepressed patients, and roughly 50% with a viral load >5 log10 HIV-RNA copies/ml, the likehood of virological failure was low, accounting for 2.8% and 4.3% by the first year in those presenting with CD4 counts less than 350 and less than 200/mma cohort .3 or AIDS. Nevertheless, looking at more advanced patients, with CD4 counts up to 200 cells/mm3 or AIDS, LPV/r including regimens resulted to be at lower risk of virological failure as in the multivariate model, ATV/r showed a 3.7-fold statistically significant higher risk and DRV/r a 3.1-fold not significant higher risk.In this perspective, we did not found any differences in adjusted risk of virological failure according to the PI/r given in those patients starting cART with CD4 counts less than or equal to 350 cells/mmLooking at the other endpoints, i.e. PI/r discontinuation for any causes or for toxicity and treatment failure, we did not find different predictors according to the severity of immune depression: the risk of discontinuing the PI/r of the regimen for any causes was higher for patients given LPV/r as compared the other two PI/r, after adjustment for other possible predictors; limiting the end-point to those discontinuing only because of toxicity, there were no differences in risk of this event when comparing ATV/r to LPV/r, while patients given DRV/r including regimen were at lower risk of discontinuation for toxicity. Finally, LPV/r including regimens were at higher risk of treatment failure as compared to the other PI/r regimens, in both the groups of immune depressed patients.To date there is no single trial comparing the different PI/r regimens in the population of severely immune depressed patients. Actually, the head to head Castle trial  comparinThe Artemis trial  was desiFinally the ACTG5257 trial  included3, LPV/r including regimen were less virologically potent than ATV/r ones. This last finding is not confirmed by our data in an observational setting, highlighting that LPV/r including regimens resulted the more potent than both ATV/r and DRV/r (even if in this last case not significantly different) in subjects with very low (< = 200/cmm) CD4 counts or AIDS.Taken together, we can argue that overall there was no major difference in virological potency across the three PI/r in randomized controlled trials, but in case of patients with CD4 counts <200/mmObservational data may be biased for many reasons, but they are more adherent to real life than clinical trials. Possibly, LPV/r including regimens, to be administered twice a day and with a double boosted ritonavir, could be more potent that the other PI/r, as forgiveness could be less important.Looking at discontinuation, LPV/r including regimens resulted to be those associated to higher risk of discontinuation across both CD4 strata, confirming the data of trials –12, partAlso, the higher risk of discontinuing the PI component of the regimen was true also for ATV/r as compared to DRV/r, confirming the finding of the ACTG5257 trial , possiblIt is not surprising that, taking into account both efficacy, tolerability, scheduled timing of administration and toxicities, LPV/r including regimens are those associated to higher risk of treatment failure, accordingly to clinical trails, and actually all guidelines have downgraded LPV/r to a second-line choice in initial cART –8, even In conclusion, based on our data in a real life setting, focused on severely immunodepressed patients, the overall efficacy and durability of the PI/r including regimens is high, with a likehood of only 4% and 21% of virological failure and discontinuation by one year.New integrase including regimens, demonstrated to be at least at similar virological potency, but with less toxicities than PI/r in the clinical trials , 16–17 sS1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This retrospective study aimed to investigate that if switch of combination antiretroviral therapy (cART) would result in viral suppression (<40 copies/mL) at 48 weeks for patients with persistent low-level viremia after having received cART for six months or more at two hospitals designated for HIV care in Taiwan.Between January 2001 and January 2013, patients were enrolled if plasma HIV RNA load (PVL) were >20 to <1000 copies/mL detected for six months or more [In this study, 165 patients were enrolled, 119 patients (72.1%) did not switch (Group 1), and 46 patients (27.9%) switched previous regimens to ARV of different mechanism (Group 2). The two groups differed significantly in the proportion of injecting drug users (IDU)  and median PVL (67 vs 159 copies/mL), and the proportion of PVL<200 copies/mL (84.0% vs 58.7%) when low-level viremia was first detected. In Group 1, 39 (32.8%) continued two nucleoside reverse-transcriptase inhibitors (NRTIs) plus nNRTI; 29 (24.4%) 2 NRTIs plus PI, 47 (39.5%) 2 NRTIs plus boosted PI, and 4 (3.3%) 2 NRTIs plus integrase inhibitor (II). In Group 2, two (4.3%) switched to 2 NRTIs plus PI, 38 (82.6%) 2 NRTIs plus boosted PI, three (6.5%) 2 NRTIs plus II and three (6.5%) boosted PI plus II. In multivariate analysis, IDUs  and PVL of 200–999 copies/mL at enrollment  were more likely to be switched. At 48 weeks, patients in Group 2 were more likely to achieve PVL<40 copies/mL than Group 1 , while no difference was observed in achieving PVL <200 copies/mL between the two groups . According to the clinical guidelines of BHIVA, patients with low-level viremia who switched to cART consisting of 2 NRTIs plus boosted PI or newer mechanisms were more likely to re-establish viral suppression to <40 copies/mL at week 48.


In our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at previous failures to a Raltegravir containing regimen, in patients who are currently undetectable and candidates to simplification to a Dolutegravir containing regimen, in order to decide on once a day or twice a day dosing.We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29–53) Integrase resistance mutations in proviral DNA were studied.All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was of 1.3 log cp/ml (range 0–1.47) and 765.5 cells/μL . The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29–53) months. At Raltegravir failure, N155H was detected in four patients, and other secondary mutations were detected in five patients (71.4 %). In proviral DNA, N155H was detected by population sequencing in three patients (42.8 %), and UDS demonstrated a 9.77 % relative abundance of N155H in the remaining patient. Sanger sequencing correctly identified all the secondary mutations.This is a pilot study that demonstrates the possibility of properly identifying N155H and some secondary mutations 29–53 months after failure. For the last 6 years, Raltegravir (RAL), the first strand Integrase transfer inhibitor (INIs) approved, has played a relevant role on the treatment of HIV infection, both for naive and pretreated patients .Nowadays, Elvitegravir (EVG) and Dolutegravir (DTG), two new INIs have become available. EVG is a first generation drug and shares its resistance profile with RAL, whereas DTG is a second generation INI with a high antiviral effect, and excellent efficacy and safety profile. Although DTG has a high genetic barrier to resistance, its activity may be limited by certain combination of resistance mutations that may accumulate during failure to RAL . The ocuIn our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at failure to RAL, and we attempted to provide the proof of concept that testing proviral DNA may be used as a sentinel for RAL mutations, in patients who are currently undetectable and candidates to simplification to a DTG containing regimen.We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29–53), Integrase resistance mutations in proviral DNA were studied.6 leukocytes aliquots, that were used for DNA extraction with QIAamp DSP DNA Blood Mini Kit (QIAGEN). DNA was quantified by spectrophotometry .Peripheral Blood Mononuclear Cells (PBMC) were separated by Ficoll gradient centrifugation, washed, counted and pelleted to 5x10Integrase was amplified using a nested-PCR. For the first round of amplification, we used outer primers IN1F (5′-GGAAAAGGTCTACCTGTCATGGGT-3′) and IN1R (5′- GGAGAAAGAGACTGGCATTTGG-3′) and the following PCR profile: 94 °C/2′;  x35 cycles; 72 °C/7′; 4 °C. This profile was used for the nested PCR, using inner primers INR2F (5′- TGGAGGAAATGAACAAGTAGATAAATT-3′) and (INT2R: 5′-GGGTCTGCATACAGGAGAAA-3′).For Sanger sequencing, we used a bidirectional sequencing protocol and the Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit (Affymetrix USB), which was optimized for the TruGene/OpenGene DNA sequencing system (SIEMENS); sequencing primers were P1F:5′- Cy5.5-GTAGCCAGCTGTGATAAATGTC-3′) and P2R (5′- Cy5-CTGCCATTTGTACTGCTGTCT-3′), allowing to sequence 414 nucleotides in the Integrase region, covering positions 40 to 178. The amplification profile was: 94 °C/5′;  x30 cicles; 70 °C/5′; 4 °C. The obtained sequences were aligned and combined using OpenGene Geneobjects™ software, and fasta sequences were interpreted Stanford HIV Database Versión 7.0 [Ultra Deep Sequencing (UDS) was done using an HIV-1 UDS prototype (Roche diagnostics) and the 454 GS Junior . As template for the UDS PCR, we used the same amplification products as for standard Sanger sequencing following from this point the manufacturer’s recommendations. Once UDS was performed, sequences were exported by AVA  software and interpreted by DeepChek®- VIH  software, using for interpretation the same Stanford version as for Sanger sequencing.This was a retrospective, non-interventional study, and patient information was anonymized and de-identified prior to analysis.All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was 1.3 log cp/ml (range 0–1.47) and 765.5 cells/μL . The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29–53) months. Detailed information on baseline characteristics is shown in Table Table Dolutegravir has shown excellent efficacy and safety in individuals infected by HIV both in naïve , and patProviral DNA may be a useful tool to investigate the presence of resistance mutations –14, espeIn our study, using Sanger sequencing of the Integrase region of proviral DNA, we could correctly identify failing selected mutations in 6/7 patients. Although for the remaining patient we could not demonstrate the failing mutation with Sanger sequencing, using a more sensitive test , resulteAlthough the sampling time in patients 2, 3 & 7 exceeded the half-life of the HIV-1 reservoir, this did not interfere in the correlation between the failing sample and proviral DNA testing. Despite some studies have demonstrated that the latent viral reservoirs half-life is from four to six months in patients who start therapy in the acute infection stage, there are other studies in chronically infected patients who have shown a half-time of 44 months , 17.Our study has certain limitations. First, only subtype B patients have been included, so the methodology needs to be validated for other subtypes. Secondly, only the N155H pathway was confirmed in some patients and it is possible that resistance pathways other than N155H, that could have emerged before N155H was established, may have been archived in the proviral DNA of the patients compromising DTG activity.In summary, despite the limitations of our study, which is just a pilot study that should be confirmed in further studies, we have shown the proof of concept that for patients who failed a Raltegravir containing regimen in the past, who are currently virologically suppressed, and lack the resistance information at failure, studying Integrase resistance in the proviral DNA accurately reflects the possibility of properly identifying N155H and some secondary mutations 29–53 months after failure.The Ethics Committee of the San Cecilio Hospital approved the study, and no consent information was required as patient information was anonymised and de-identified prior to analyses.Not applicable.All data supporting our findings is contained within the manuscript.


Short-medium term studies from sub-Saharan Africa show that, despite high early mortality, substantial loss to program, and high rates toxicity, patients on antiretroviral treatment have achieved outcomes comparable to those in developed settings. However, these studies were unable to account for long term outcomes of patients as they stayed longer on treatment.We aim to describe ten years outcomes of one of the first cohort of HIV positive patients started on antiretroviral treatment (ART) in Sub-Saharan Africa.We report 10-years outcomes including mortality, retention, CD4-count response, virological outcomes, ART regimens change from a prospective cohort of 559 patients initiating ART and followed up for 10 years Uganda.Of 559 patients, 69.1% were female, median age (IQR) was 38 (33–44) years, median CD4-count (IQR) 98 (21–163) cell/μL; 74% were started on stavudine, lamivudine and nevirapine, 26% on zidovudine, lamivudine and efavirenz. After 10 years 361 (65%) patients were still in the study; 127 (22.7%) had died; 30 (5%) were lost to follow-up; 27 (5%) transferred; 18 (3%) withdrew consent. The probability of death was high in the first year . The median CD4 count increased from 98 to 589 cell/μL (IQR: 450–739 cell/μL) with a median increase of 357 cells/μL (IQR: 128–600 cells/μL); 7.4% never attained initial viral suppression and of those who did 31.7% experienced viral failure. Three hundred and two patients had at least one drug substitution while on first line after a median of 40 months; 66 (11.9%) of the patients were switched to a second line PI-based regimen due to confirmed treatment failure.Despite the high rate of early mortality due to advanced disease at presentation the outcomes from this cohort are encouraging, particularly the remarkable and incremental immune-recovery and a satisfactory rate of virologic suppression. Over 7.6 million individuals are receiving antiretroviral therapy (ART) in sub-Saharan Africa (SSA) as a result of unprecedented global health initiatives to scale-up ART delivery since 2003. Uganda Short and medium term studies from sub-Saharan Africa show that despite high early mortality rates due to late presentation, substanThe Infectious Diseases Institute at the Makerere University College of Health Sciences in Kampala was one of the first HIV care centers to receive free antiretroviral drugs from GFATM and PEPFAR, therefore some of the active patients attending the adult clinic have been followed up for more than ten years. We have previously described the short term (one-year) and medium term (four-years) outcomes from a well characterized ART cohort within a large urban HIV treatment center at the National Referral Hospital in Uganda . The objThe study was reviewed and approved by the Makerere University Faculty of Medicine Research and Ethics Committee  and the Uganda National Council for Science and Technology . All patients provided written informed consent.The Infectious Diseases Institute (IDI) is a large urban HIV center of excellence  located In this context a cohort of consecutive adult patients starting ART was initiated and 559 patients were enrolled in a research cohort between April 2004 and April 2005. The main objective of this study, hence forth referred to as the IDI Research Cohort, was to assemble and to characterize a cohort of patients on ART in order to address pertinent epidemiologic, clinical and psychosocial research questions relevant to the national and sub-Saharan African ART roll-out.At enrollment and every three months patients were evaluated by a dedicated study clinician and counselor. ART was started according to WHO and Uganda Ministry of Health guidelines ,14 in paDetailed study procedures for this is cohort are described elsewhere, but in Laboratory investigations were performed every 6 months and these included complete blood cell count, liver and renal function tests, CD4 count by FACSCount , HIV viral load by Amplicor HIV-1 Monitor PCR Test version 1.5 and more recently, COBAS Ampliprep/COBAS Taqman HIV-1 Test Ver.2.0  and storage of 5 ml of plasma at -80 C for future testing. Laboratory testing was performed at the Makerere University–Johns Hopkins University Core Laboratory, which is certified by the College of American Pathologists.Tracking and complete ascertainment of patients who did not attend their study appointment was carried out in real time by phone call and/or home visit. In case of death the cause was determined from a combination of source documents and a structured interview with the patient’s next of kin . Generally ascertainment of outcomes of nearly all study subjects was achieved and lost to follow up was negligible.Data was collected on a study specific designed questionnaire by the study counselor and the study clinician. The data was entered into a database designed using Oracle up to year 2011 and in ICEA (Integrated Clinic Enterprise Application), an application developed by a team of software developers based at the IDI, which ensures good quality of collected data .The entered data was subsequently validated by a senior data entrant before being approved. Laboratory results were obtained directly from the laboratory database. The data was also regularly checked for quality, completeness and discrepancies by a data quality assurance team and a data manager.retained in the cohort if he/she was alive and still enrolled in the cohort. A patient was defined as lost to follow up if two consecutive study visits were missed, i.e. the patient did not attend the clinic for more than 6 months.A patient was considered Viral suppression was defined as the attainment of HIV viral load <400 copies/ml after starting ART in patients with at least 6 months of follow up, and in those with documented viral suppression, viral failure was defined as 2 consecutive viral load measurement >400 copies/ml or 1 viral load> 5,000 copies/ml if a subsequent viral load was not available. Treatment failure was defined as the lack of attainment of viral suppression or the occurrence of viral failure after initial suppression.Treatment change was defined as the occurrence of drug substitution or regimen switch. Drug substitution was defined as the modification of at least one drug within first line regimen, while regimen switch was defined as a change from a first to a second line regimen.Baseline characteristics were described using medians and proportions and stratified by gender. Gender categories were compared using chi-square test for categorical variables and Wilcoxon rank-sum test for not normally distributed variables.To describe retention in the cohort we reported the number and proportion of patients who were no longer enrolled in the cohort for any reason including loss to follow up, death, transfer out, and withdrawal of consent at month 6, 12, and year 2, 5 and 10. We reported mortality over a 10-year time period by cause: HIV related, other medical, and accidental and we calculated the probability of death using Kaplan Meier estimates with right censoring at time of death, stratified by gender and CD4 count μL at ART start .We described the increase in CD4 count from baseline to all subsequent 6-monthly CD4 count readings and the overall increase from baseline to the last visit in the cohort. We also reported the proportion of patients at each time point with a CD4 count >400 cells/μL, which is the lower normal limit of CD4 count in Ugandan adults .We reported the proportion of patients who attained viral suppression and of those who experienced viral failure. We also calculated the cumulative probability of treatment failure stratified by gender and CD4 count at ART start at different thresholds.We described the ART regimens prescribed during the 10 years categorized as stavudine, zidovudine tenofovir based for first line regimens, and PI based. We described all causes of drug substitution categorized as toxicity, treatment failure, new episode of tuberculosis, pregnancy/intended pregnancy and others, and we calculated the yearly and cumulative probability of first drug substitution for any reason while of first line.All differences in survival probabilities were compared using the log rank test.The analysis was performed using STATA® version 12.2, Texas USA.Between April 2004 and April 2005, 559 consecutive patients starting first line antiretroviral treatment were enrolled in the IDI Research Cohort. The baseline characteristics and the characteristics by gender are summarized and compared in The median follow up time was 110 months (IQR: 25–111). The probability of death was 0.15 (95%CI: 0.12–0.18) by year 1, 0.21 (95%CI: 0.18–0.25) by year 5, and 0.24 (95%CI: 0.21–0.28) by 10 years on ART. The majority  of the deaths were AIDS events, followed by other medical condition  and 1 (0.8%) accidental death; the cause of death could not be determined for 16 (12.6%) patients due to lack of relative contact or non-conclusive “verbal autopsy”. Patients with a CD4 count <50 cells/μL at ART start had a significant higher overall probability of dying, 0.31 (95%CI: 0.25–0.39) as compared to patients with a CD4 count ≥100 cells/μL, 0.18: (95%CI: 0.14–0.23) . We did not find an independent association between survival and gender .The median CD4 count increased from 98 to 589 cell/μL (IQR: 450–739 cell/μL) over the 10 year period with a median increase of 357 cells/μL (IQR: 128–600 cells/μL). The proportion of patients with a CD4 count >400 cells/μL rose from 0.9% at baseline to 83.8% after ten years on ART, with majority of patients (50.9%) being above this threshold by year 5 on ART . The cumFour hundred seventy four patients were followed up for at least 6 months, and of these 35 (7.4%) never attained viral suppression; the cumulative probability of attaining viral suppression was 0.94 (95% CI: 0.92–0.96). Among the 439 (92.6%) patients who attained viral suppression 139 (31.7%) experienced subsequent viral failure with a cumulative probability of 0.34 (95%CI: 0.30–0.39). Five of those who never attained viral suppression, and 14 and those who experienced viral failure subsequently died.Overall the median time to treatment failure was 30 months (IQR 11–66) and the cumulative probability of treatment failure over 10 years was 0.38 (0.34–0.43).The probability of treatment failure was similar in males as compared to females , P = 0.08) and we did not find any difference in the probability of treatment failure across the CD4 count strata at ART start (P = 0.52)th year of follow up 17.1% of the retained patients were switched to second line PI-based regimens.Three hundred and two patients (54%) had at least one drug substitution while on first line for a total of 378 drug substitutions. Two hindered forty four (64.6%) had at least one drug substitution, 46 (24.3%) two substitution and the remaining ones up to 5 substitutions. The most common reason  for the first drug substitution was the 2008 Ministry of Health recommendation to replace stavudine with a less toxic nucleoside reverse transcriptase inhibitor in patients still receiving it, followed by ART toxicity , occurrence of a new episode of tuberculosis , pregnancy , and other reasons (8. 2.6%).The median time to the first drug substitution was 40 months (20–44); the cumulative probability of drug substitution was 0.77 (CI: 0.72–0.81) with a higher probability of substitution in females  as compared to males  .Overall 66 (11.9%) of the patients were switched to a second line PI-based regimen due to confirmed treatment failure.To our knowledge this is the first comprehensive analysis of 10-year outcomes in a well characterized African ART cohort. We observed that despite a high early mortality, the overall retention and viral suppression rates were high with remarkable immunologic recovery over the 10 year period. These findings are encouraging given the context of an initially highly immune-deficient population. Unfortunately this success was overshadowed by high rates of early mortality due to late presentation and high rate of toxicity resulting in drug substitution.Overall we observed that our cohort consisted of young, predominantly female and severely immunosuppressed individuals, representative of African ART patients at the start of the ART roll-out. The population in this cohort started ART at very advanced stage of disease, with 89% of the patients being in 3 or 4 WHO stages, and with a quarter of them having a CD4 count <21 cells/μL. Male patients were generally older and had a lower BMI probably due to a later presentation into HIV care.The overall rate of loss to follow up was very low, but this may not reflect the attrition rates observed in other settings , or evenDespite a low baseline CD4 count, we report remarkable immune recovery (median CD4 count after 10 years: 589 cells/mL) with CD4 count levels continuously increasing throughout the 10 years follow up period up and with 83% of the patients attaining a CD4 count above the lower normal limit. The reduction in incidence of OIs including tuberculosis (data not shown) and mortality is to a large extent attributable to this immune recovery. In this analysis we included all patients regardless of viral suppression and ART regimen in order to demonstrate the general effect of ART on population immune recovery over time.We also observed a high initial viral response comparable or higher than observed in on treatment analysis from similar African  and non-Similarly to other reports of patients started on ART at the beginning of the scale up ,22, a hiMore than half of the patients required at least one drug substitution while on first line; this was mostly due to the inclusion of stavudine in the majority of the ART regimens prescribed at treatment start. Most of the substitutions due to toxicity were attributable to stavudine toxicity  while thIn summary, although the success of ART was jeopardized in the early years by early mortality and ART toxicity, the outcomes from this cohort are encouraging, particularly the remarkable and incremental immune-recovery and a satisfactory rate of virologic suppression. Patients presenting with less advanced disease and those started on ART later during the scale up when stavudine was no longer part of the preferred regimens are likely to experience even more favorable outcomes.


Patients receiving antiretroviral therapy (ART) require routine monitoring to track response to treatment and assess for treatment failure. This study aims to identify gaps in monitoring practices in Kenya and Uganda.We conducted a systematic retrospective chart review of adults who initiated ART between 2007 and 2012. We assessed the availability of baseline measurements  and ongoing CD4 and weight monitoring according to national guidelines in place at the time. Mixed-effects logistic regression models were used to analyze facility and patient factors associated with meeting monitoring guidelines.From 2007 to 2012, at least 88% of patients per year in Uganda had a recorded weight at initiation, while in Kenya there was a notable increase from 69% to 90%. Patients with a documented baseline CD4 count increased from 69% to about 80% in both countries. In 2012, 83% and 86% of established patients received the recommended quarterly weight monitoring in Kenya and Uganda, respectively, while semiannual CD4 monitoring was less common (49% in Kenya and 38% in Uganda). Initiating at a more advanced WHO stage was associated with a lower odds of baseline CD4 testing. On-site CD4 analysis capacity was associated with increased odds of CD4 testing at baseline and in the future.Substantial gaps were noted in ongoing CD4 monitoring of patients on ART. Although guidelines have since changed, limited laboratory capacity is likely to remain a significant issue in monitoring patients on ART, with important implications for ensuring quality care. Over the past decade there has been a massive scale-up of antiretroviral therapy (ART) in sub-Saharan Africa. Between 2003 and 2012, the number of people receiving ART in the region rose from 100,000 to 7.5 million ,5. With At the time of ART initiation, proper evaluation can identify severely underweight patients, as well as those with very low CD4 counts who are at higher risk of poor outcomes and may require closer clinic and provider follow-up ,7. FurthOnce initiated, monitoring a patient’s response to therapy allows providers to detect potential adherence problems and treatment failure. It is critical to promptly identify treatment failure, as switching to a second-line regimen for these patients is essential for reducing mortality risk and preventing the spread of drug resistance –11. UntiDespite the need for risk stratification and ongoing monitoring while on ART, relatively little is known about how these guidelines are followed in practice. Laboratory testing in sub-Saharan Africa has largely been hindered by inadequate infrastructure, equipment, and reagent shortages, and low availability of skilled laboratory professionals ,22. AddiAs Kenya and Uganda pursue new monitoring guidelines that include regular viral load testing, it is important to understand how well prior guidelines were followed and to consider why they may have fallen short in practice. In this paper, we assess recent CD4 and weight monitoring practices and examine facility- and patient-level characteristics associated with meeting national monitoring guidelines.This study used data from a subset of facilities from a larger, multi-country facility-based project that took place in Kenya and Uganda  . NationaHealth facilities that declined study participation or where access to the facility was limited due to safety, travel distance, or time constraints were replaced with other similar facilities within the same district by the country team when a suitable replacement facility was identified. Data collection took place from April to November 2012 in both countries.At all selected facilities that provided ART services, we conducted a retrospective chart review of adult patients (18 years and older) who initiated ART 6 to 60 months prior to the survey date. In addition to patients actively in care, we sought to include the charts of all transferred, defaulted, and deceased patients. We received electronic medical records for all patients meeting inclusion criteria at four facilities in Kenya. At the remaining facilities in both countries, the facility administrator reported the total number of eligible charts, and we sampled up to 250 charts using an equal-probability procedure. At facilities with less than 250 charts, we included all charts in our study. Two facilities in Uganda provided electronic records for the sampled patients, while all other information was extracted from paper charts.Trained research associates interviewed facility administrators to collect information about facility management, resources, practices, and patient volumes. They then extracted information related to patient demographics, initiation characteristics, treatment regimen, outcomes, full visit history, and all test results  from the sampled ART charts. Research associates also searched each patient’s folder for documentation of weight, CD4 count, or viral load tests that were not recorded in the standard chart and extracted the relevant information.We applied sample weights based on the reported number of adult patients in each facility’s ART program, such that our reported values are representative of all patients at the sampled facilities. Analyzes related to initiation were weighted based on the annual number of new initiates, while analyzes for ongoing monitoring used the annual number of enrolled patients. We linearly extrapolated patient numbers for the 9% of facility-years where these data were not reported. We also extrapolated patient numbers for all facilities in 2012 as the facility survey only collected information through 2011.We examined whether each patient met existing national guidelines for minimum monitoring at baseline and once they were established on therapy. These guidelines were consistent across countries and years for our study period (2007–2012) ,16,30.At ART initiation, both countries called for a measure of weight, CD4 cell count, and WHO stage. We determined whether each measurement was recorded at some time between three months prior to and one month following initiation to account for lags between eligibility testing and actual ART initiation; this approach also accounted for the potential that tests performed at ART initiation could be reviewed and recorded at a subsequent visit.established, meaning they initiated ART at least six months before the given month, and (b) retained, meaning they were alive and on treatment during the given month, and not transferred, dead, or defaulted . A patient met CD4 testing guidelines for a given month if a CD4 test was recorded at any point in the prior six months. Likewise, they met weight guidelines if a weight measure was recorded in the prior three months. If a patient did not have a test recorded during a specified time period, we determined whether the patient visited the facility during that period without receiving a test. Results for a given facility type-country-month are only shown if at least 50 charts were included in the calculation.After the initiation period  monitoring guidelines called for a weight measurement every three months and CD4 measurement every six months, at minimum ,16,30. FWe used a series of mixed-effects logistic regressions to assess factors associated with meeting minimum guidelines. For each country, models were run at the patient level using facility random effects.Two separate analyzes were performed. The first examined determinants of having a baseline CD4 test and weight measurement, run as separate models, among patients who initiated therapy between 2011 and 2012. This group, the mostly recently initiating patients in our sample, was selected because determinants of testing may have changed over time and more recently measured factors are most relevant to policy and practice.The second set of analyzes examined the extent to which established patients were meeting monitoring guidelines at the time of the survey. Patients were included in this analysis if (1) they were alive and in care at the time of the survey rather than transferred, dead, or defaulted; and (2) they had initiated at least 12 months prior to the survey, to ensure results were not influenced by elevated testing in the period immediately following initiation. Separate models examined two binary dependent variables: (1) CD4 test recorded during the six months prior to record extraction; and (2) weight recorded in the three months prior to record extraction.a priori based on potential theoretical relationships with monitoring practices. All models included patient-level characteristics (age and sex), as well facility-level indicators including facility type ; ownership (public or private); age of ART program; on-site CD4 analysis capacity; receipt of HIV-specific staff training during the past year; and whether nurses led patient treatment.Independent variables were selected Models assessing baseline CD4 and weight measurements included an indicator of baseline disease severity as categorized by four WHO stages.Models examining recent measurements for established patients included duration on ART and binary indicators capturing whether the patient showed signs of WHO-defined immunological or clinical failure in the six months preceding the testing window of interest. Per WHO definitions , potentiAll analyzes were conducted in Stata 13.1 .Ethical approval for this study was obtained from the University of Washington Human Subjects Division and local institutional review boards in Kenya  and Uganda (Makerere University School of Medicine Research Ethics Committee). Patient consent for review of clinical charts was not obtained, as all information extracted from clinical charts was anonymized and de-identified prior to analysis.Data from 23,618 patient charts were extracted from 97 facilities offering ART . The sampled facilities were largely publicly-owned (79%) and urban (67%). Most facilities had HIV guidelines available (97%), but only 26% had staff with HIV-specific training during the previous year. Ugandan facilities in our sample (80%) were more likely to have on-site CD4 analysis capacity than those in Kenya (29%). Most facilities without CD4 analysis capacity took samples on-site and shipped them elsewhere for analysis; only 3% did not offer any CD4 testing. One hospital each in Uganda and Kenya reported capacity to run viral load assays. Functional adult weight measurement scales (99%) and measuring tapes (92%) were nearly universally available.The majority of patients were female (64%) with a median age of 36 at initiation. When recorded, the median baseline CD4 count was 175 and 41% of patients were classified as WHO stage 3 or 4. Over half of patients initiated in 2010 or 2011.Between 2007 and 2012, at least 88% of patients had a WHO stage recorded at initiation across country-years . Weight In 2012, 83% and 86% of established patients met the recommended quarterly weight monitoring in Kenya and Uganda, respectively . This waIn 2012, 49% and 38% of established ART patients met the guideline for semi-annual CD4 tests in Kenya and Uganda, respectively . This fiControlling for patient and facility characteristics, a more advanced baseline WHO stage was associated with lower odds of baseline weight testing in Kenya  and lower odds of baseline CD4 testing in both countries  .Having on-site CD4 analysis capacity was associated with increased odds of baseline CD4 testing in Kenya  and Uganda . Compared to patients receiving care at hospitals, patients at urban health centers had increased odds of receiving a baseline CD4 test in Kenya  and those at rural health centers had reduced odds in Uganda . Kenyan patients at facilities with staff who received HIV training in the past year had an increased odds of a baseline CD4 test ; by contrast, receipt of HIV-specific training in Uganda was associated with a lower odds . Ugandan patients at nurse-led programs had an increased odds for the receipt of baseline CD4 testing .In comparison with patients receiving ART at hospitals, Ugandan patients at urban health centers had an increased odds of meeting weight guidelines  and those at rural health centers had a reduced odds of meeting CD4 guidelines . Patients at urban health centers had increased odds of meeting CD4 testing guidelines in Kenya .Kenyan patients had an increased odds of receiving a CD4 test in the past six months if they received care at a facility with staff who received HIV-specific training during the last year . In Uganda, such training was associated with an increased odds for patients meeting weight guidelines , but a reduced odds for meeting CD4 testing guidelines . In Uganda, patients who received care at a HIV program with nurse-led care also had increased odds of meeting CD4 testing guidelines .For established patients in Uganda, prior indications of immunological failure were associated with an increased odds of meeting weight monitoring guidelines , while a previous indication of clinical failure was associated with an increased odds of meeting the semi-annual CD4 testing guidelines  .Kenya and Uganda have both made tremendous gains in enrolling patients on ART. However, in the setting of increased financial constraints and attention towards quality of care, a careful examination of monitoring practices is essential. As monitoring guidelines for ART patients transition to include viral load testing, our findings raise relevant questions about clinic visit frequency, laboratory capacity, and adherence to international and national norms.We found that patients were visiting the clinic quite frequently, and on average 90% of established patients had a visit every three months in 2012. Since most of these patients were likely to be stable and required little or no intervention, it is important to consider whether quarterly clinic visits for this established stable population on ART is necessary, as a reduction in visit frequency could have a significant impact on facility costs and efficiency . ReducinOur study found that most ART patients in our sample regularly visited facilities, suggesting that patient monitoring may be primarily limited by facility resources and actions rather than care-seeking behaviours. While low-tech, point-of-care weight monitoring was feasible and performed according to guidelines at most facilities, less than 50% of patients in care received a CD4 test during the previous six months in both Kenya and Uganda. Given that ART patient volumes dramatically increased during the study period, the overall number of CD4 tests has undoubtedly grown; at the same time, facilities barely kept pace or fell behind in the proportion of their patients receiving CD4 tests for continuous monitoring. In Uganda, we found that facilities where staff HIV training was performed within the prior year the odds of having a CD4 at baseline or afterwards was lower than in facilities where there was no such training. This could potentially be explained by an even more dramatic increase in patient volumes in facilities with additional training/expertise leading to demand for testing outstripping testing capacity. Alternatively, facilities where HIV training was performed may have noted less benefit in recurrent CD4 counts, and reserved testing for patients who appeared clinically ill or had another indication for CD4 testing. We also found that CD4 testing was strongly related to having on-site analysis capacity. As CD4 testing remains one of the primary means for determining ART initiation, our results support equipping facilities to analyze CD4 tests, or at a minimum improving systems through which off-site CD4 test analysis can occur efficiently and at low cost. Our findings complement previous research on the benefits of point-of-care CD4 testing to reduce attrition between testing and initiation ,35.Experiences with off-site CD4 analysis are also highly relevant as countries begin scaling up viral load monitoring. While viral load testing offers benefits over immunological and clinical monitoring , this guOur findings should be viewed in light of a number of limitations. First, we only examined tests and visits recorded in patient charts. While these data reflect the information available to providers for monitoring purposes, we cannot quantify how much record-keeping practices affected our results concerning gaps between recommended and observed testing practices. Second, patient charts did not contain demographic characteristics, such as educational levels and household wealth, which could be important determinants of health behaviors. Third, chart storage for deceased or defaulted patients may vary across facilities, a practice that could introduce biases. Further, electronic medical records received from six facilities may differ from the paper records used at the remaining facilities. At the two Ugandan facilities with electronic records, 100% of patients received a CD4 test at initiation. Since we sampled only a few facilities with electronic records, we are unable to determine if this relationship was due to record-keeping procedures, overall managerial and financial capacity, or a causal relationship between electronic records and better monitoring.5 Future research will need to address the frequency of routine clinic visits and testing, while national HIV treatment programs will also need to look toward improving current laboratory capacity. Striking this balance in terms of patient visit quantity and quality is critical to ensuring that patients receive the maximum benefits of ART.Despite these limitations, our study identifies a clear gap between prior immunological monitoring guidelines and clinical practice. These findings are particularly relevant as the number of patients eligible for ART continues to increase due to changing initiation guidelines.


TDF/FTC/RPV has been shown effective in both naïve and PI-pre-treated patients. Less is known about a switch strategy in subjects receiving EFV.We evaluated viro-immunologic outcomes, Quality of Life (QoL) and costs of an unselected cohort of patients switching from a TDF/FTC/EFV STR (≥6 months duration) to a TDF/FTC/RPV STR. The considered outcome measures were quality-adjusted life years (QALYs) as measured with the EQ5D questionnaire and the overall direct health costs. 64 patients with a baseline viral load<50 copies/mL were randomized to immediately switch therapy or to continue TDF/FTC/EFV for four months and then switch to TDF/FTC/RPV. Six patients in the deferred switch group did not actually change cART.Patients were mostly males (73.4%) with a mean age of 46 years, a baseline mean HIV-RNA of 6.4 copies/mL and a mean baseline CD4 count of 588 cells/µL. For the considered follow-up period, the mean cost per patient resulted 2,563 for TDF/FTC/RPV and 2,572 for TDF/FTC/EFV. Viremia remained undetectable and CD4 stable in all patients. Over time the mean QoL increased in the RPV arm ad slightly decreased in the EFV arm, after four months the mean per patient QALYs was 0.849 for RPV and 0.841 for EFV, respectively . A sharpSwitching from TDF/FTC/EFV to TDF/FTC/RPV is a safe, well tolerated strategy that improves the overall health status of HIV-treated patients. The switch does not expose patients to a risk of virologic failure due to possible PK interactions of the drugs. RPV compared to EFV proved to be cost-effective showing lower cost and higher outcome measure values.


We investigate whether this intervention affects viral suppression and CD4Longitudinal observational study.We identified adult patients from Concerted Action of Seroconversion to AIDS and Death in Europe who seroconverted after 1/1/2000, had a 12 months or less HIV test interval and initiated cART from naive. We classified individuals as ‘pretreated in EHI’ if treated within 6 months of seroconversion, interrupted for at least 12 weeks, and reinitiated during CHI. Statistical analysis was performed using survival analysis methods and mixed models.P = 0.585 and P = 0.206) but pretreated individuals restarted treatment with higher baseline CD4+ cell count  and retained significantly higher CD4+ cell count for more than 3 years after treatment (re)initiation. Assuming common baseline CD4+ cell count, differences in CD4+ cell count slopes were nonsignificant. Immunovirologic response to CHI treatment was not associated with timing or duration of the transient treatment.Pretreated and initiated in CHI groups comprised 202 and 4263 individuals, with median follow-up after CHI treatment 4.5 and 3 years, respectively. Both groups had similar virologic response and relapse rates (Although treatment interruptions are not recommended, stopping cART initiated in EHI does not seem to reduce the chance of a successful outcome of treatment in CHI. Out of 184 pretreated individuals with viral load measurements available during their EHI transient treatment, 133 (72%) had less than 50 and 29 (16%) 50–500 HIV-RNA copies/ml before interrupting treatment.Estimation of the seroconversion date among pretreated individuals was mostly accurate as 65.8% of them had a seroconversion illness, laboratory evidence of seroconversion or an HIV test interval of less than a month Table . Among tTiming and frequency of viral load measurements after cART initiation in CHI were similar in the two groups, with the first measurement taken after a median (IQR) of 1.2  months.The distribution of viral loads at different time points after CHI-cART initiation is summarized in Fig. The estimated cumulative probabilities of achieving less than 50 copies/ml viral load are presented in Fig. P = 0.585). Estimated acceleration factors were greater than 1 for all other risk groups compared to MSM, indicating longer average times to achieve viral suppression. A clear trend for higher rates of virologic response to treatment initiated in more recent years was also evident. Higher initial viral load levels were associated with longer times to virologic response. Finally, regimens based on NNRTIs were associated with better performance as the average times to achieve viral load undetectability were shorter compared with those associated with PI-based regimens.Multivariable analysis Table  did not P = 0.206). Relapse rates were higher in men, injecting drug users, those treated in earlier years and those (re)starting treatment with very low viral load.Approximately 20% of those who achieved virologic suppression  relapsed by the 24th month after the initial virologic response Fig. c. The esP = 0.624 and P = 0.581, respectively). Similarly, there was no evidence of an effect of the cART duration during EHI .We also found no evidence to suggest that virologic response or relapse following reinitiation in CHI were associated with the interval between estimated seroconversion and cART initiation in EHI (+ cell count measurement post CHI-cART initiation was taken after a median (IQR) of 1.3  months with no significant difference between the two groups.The first CD4+ cell count by time and group is summarized in Fig. + cell counts. CD4+ cell count increases seem faster during the first 3 months of therapy with decelerating rates thereafter. CD4+ dynamics seem similar between the two groups with no marked differences, in terms of CD4+ cell count slope but, as in Fig. The distribution of CD4+ cell counts compared to those who were previously naive. The estimated average (95% CI) baseline CD4+ cell counts for the pretreated and previously naive individuals, for the reference category , were 460  and 383  cells/μl, respectively (P < 0.001). Individuals initiating in CHI had faster CD4+ increases in general, but the difference was significant only during months 3–30 after treatment initiation (P = 0.015). Boosted PI-based regimens were associated with faster increases during the first 3 months and injecting drug users had a worse CD4+ response, compared to men infected through sex between men throughout the whole study period. Younger individuals had faster initial CD4+ cell count increases but this association was reversed after the first 3 months of treatment. Finally, individuals of black or Hispanic ethnicity had lower CD4+ cell counts at baseline, compared with white individuals, whereas higher baseline plasma viral load levels were associated with lower baseline CD4+ cell count but faster increases after treatment initiation.Results from a multivariable mixed model are shown in Table + cell count at treatment initiation in CHI or the subsequent rates of CD4+ cell count increase (P = 0.722). The duration of the transient EHI treatment was positively associated with the baseline CD4+ cell counts at CHI treatment initiation (P = 0.020) but had no significant effect on the subsequent CD4+ slopes .The time interval between seroconversion and initiation of treatment in EHI for the pretreated group did not significantly affect the initial CD4+ cell count evolution for the reference category (as previously described) is shown in Fig. + cell count increases, compared to those who had initiated in EHI, but their CD4+ cell levels remain lower, even after 3 years of treatment . However, as pretreated individuals restart treatment in CHI at higher CD4+ cell count compared to those who were initiating in CHI, we applied a postestimation adjustment assuming individuals in both groups re(initiate) treatment at the same CD4+ cell count level . As can be seen in Fig. + cell counts compared with those pretreated during EHI, but there is a large degree of overlap in the corresponding 95% CIs, indicating that differences are not significant (P = 0.077).The average CD4+ cell count than those initiating in CHI. Even though the subsequent rate of CD4+ cell count increase was slightly slower for those who had previously been treated in EHI; they retained higher CD4+ cell counts even after 3 years of treatment. However, assuming a common baseline CD4+ cell count, the estimated difference between the two groups was not significant after 3 years of treatment. Duration of transient cART in EHI and its timing relative to estimated seroconversion did not appear to have any significant effect on virologic or immunologic outcome of long-term cART in CHI. We cannot rule out, however, that shorter cART duration in EHI would have adversely affected outcome following reinitiation in CHI.Our study assessed virologic and immunologic outcomes of long-term cART, initiated during CHI, in persons who were pretreated with transient cART during EHI compared with those who initiated in CHI. Our findings suggest that prior treatment during EHI has no effect on virologic response or relapse following reinitiation of long-term cART. Pretreated individuals reinitiated cART in CHI at approximately 80 cells/μl higher CD4+ cell counts or AIDS. Moreover, all comparisons were based on multivariable models which included adjustments for potential confounders. Finally, the study population is drawn from a number of countries , and includes individuals who had acquired HIV through both sex between men, and sex between men and women, and regimens used for long-term cART were equally based on the two most common antiretroviral classes .The main strength of our study is its size and length of follow-up. To our knowledge, this is the largest study focusing on this comparison, including 202 pretreated and 4263 previously untreated individuals with a median follow-up, after the initiation of treatment in CHI, of approximately 4.5 and 3 years, respectively. Given that this is an observational study, efforts have been made to minimise the effect of bias. For example, we excluded individuals who received cART in EHI because of low CD4Despite our restrictions regarding the time interval between the last negative and first-positive HIV test dates, a substantial proportion of individuals (mainly among those who initiated cART during chronic infection) had an HIV test window between 30 days and 1 year, allowing for some uncertainty regarding the exact time of seroconversion. It is noteworthy though that, for almost two-thirds of the pretreated individuals, the accuracy in the estimation of the seroconversion date was excellent and most of them initiated their transient treatment very soon after their seroconversion, having viral loads which were much higher compared to typical set-point levels. Thus, although we did not specifically aim to study individuals treated during acute infection, a substantial proportion of the pretreated individuals started cART very close to the time of seroconversion and this allowed us to investigate whether the gap between seroconversion and ART initiation modified our findings.+ cell counts have been excluded. Furthermore, virological failure does not appear to account for the interruption of early treatment as almost 90% of pretreated individuals interrupted with HIV-RNA less than 500 copies/ml.Additionally, reasons for starting and stopping treatment in EHI were not available. It should be noted though that the frequency of seroconversion illnesses was low, and similar in both groups, and that those who started treatment in EHI because of an AIDS diagnosis and/or low CD4+/CD8+ ratio normalization, thus our findings should be interpreted with caution.Even though differences in available factors with a significant effect on virologic and/or immunologic response were taken into account, residual confounding cannot be ruled out, as in all observational studies. Moreover, we were not able to examine differences between groups in terms of proinflammation and coagulation markers, HIV reservoir size, or CD4+ responses in long-term cART initiated in CHI with those who remained untreated during EHI. In a smaller study, Grijsen and colleagues [+ cell count response. Notably, in the same study it was found that transient treatment in EHI was not associated with clinically relevant drug resistance mutations.Our main results are in agreement with those reported by the Short Pulse Anti Retroviral Therapy at HIV Seroconversion trial investigators . As in olleagues  comparedGuidelines regarding the optimal clinical management of EHI were for many years, and partly remain, unclear –28. Howe+ cell count thresholds [+ cell count, a significant number of individuals will likely be initiating cART in EHI. We may anticipate a substantial proportion of them to interrupt, who will need to reinitiate later during chronic infection. Thus, it is clinically relevant to assess the effects of this early intervention on the outcomes of long-term treatment. According to our findings, individuals who initiate, and then stop, cART within 6 months of seroconversion do not fare differently from those who initiate in CHI, at least in terms of viral load and CD4+ cell count post-cART changes.As HIV treatment guidelines have successively recommended cART initiation at higher CD4resholds –28, and resholds  clearly Treatment interruption during CHI is associated with poorer prognosis ,36 and cFunding: The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under EuroCoord grant agreement n° 260694. K.P. is funded through a grant from the UK Medical Research Council (MC_UU_12023/1 – Infections).Authors’ contributions: Conceived and designed the experiments: N.P., G.T., and K.P. Analyzed the data: N.P. Wrote the article: N.P., G.T., and K.P. Drafted manuscript with critical revision for important intellectual content undertaken: L.M., A.O., D.C., A.D.K., I.L., M.L.C., M.F., and S.M.There are no conflicts of interest.