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INSTI-based triple regimens in treatment-naïve HIV-infected patients are associated with HIV-RNA viral load suppression at ultralow levels

INSTI-based triple regimens in treatment-naïve HIV-infected patients are associated with HIV-RNA... Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 INSTI-based triple regimens in treatment-naïve HIV-infected patients are associated with HIV-RNA viral load suppression at ultralow levels 1, 2, 3 2 2, 3 4 2, 5 Sidonie Lambert-Niclot , Anders Boyd , Djeneba Fofana , Nadia Valin , Marc Wirden , Jean-Luc 4 1, 2, 6 6 1, 2, 6 1, 2, 5 Meynard , Romain Palich , Rachid Agher , Marc-Antoine Valantin , Vincent Calvez , 1, 2, 6 1, 2, 4 1, 2, 5 Christine Katlama , Pierre-Marie Girard , Anne-Geneviève Marcelin , Laurence Morand- 1, 2, 3 Joubert Institutional affiliations: Sorbonne Université, Paris France INSERM, Institut Pierre Louis d’Epidémiologie et de Santé Publique (iPLESP), F-75012 Paris, France AP-HP, Hôpital Saint-Antoine, Laboratoire de virologie, F-75012 Paris, France AP-HP, Hôpital Saint-Antoine, Département de Maladies Infectieuses, F75012, Paris, France; AP-HP, Hôpital Pitié Salpêtrière, Laboratoire de virologie, F-75013 Paris, France ; AP-HP, Hôpital Pitié Salpêtrière, Département de Maladies Infectieuses, F75013, Paris, France; Keywords: HIV, initiation antiretroviral therapy, integrase inhibitor, residual viremia. Keypoints: In treated HIV-1-infected patients, viral load-suppression at ultralow levels is associated with INSTI-class ART. More time spent with UL-VL undetectable appears to improve immunoreconstitution and, along with CD4/CD8 ratio >0.8 over time, reduce the risk of virological rebound. © The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non- commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Corresponding author: Sidonie Lambert-Niclot, PharmD, PhD Department of Virology, Saint-Antoine Hospital 184 rue du faubourg Saint-Antoine 75571 Paris Cedex 12 Tel: +33 1 71 97 09 51 Fax: +33 1 49 28 24 82 E-mail: sidonie.lambert@aphp.fr Alternative corresponding author: Laurence Morand-Joubert,MD, PhD Department of Virology, Saint-Antoine Hospital 184 rue du faubourg Saint-Antoine 75571 Paris Cedex 12 Tel: +33 1 71 97 09 51 Fax: +33 1 49 28 24 82 E-mail: laurence.morand-joubert@aphp.fr Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 ABSTRACT Background: During antiretroviral therapy (ART), HIV-1-infected patients may present with ultralow (UL) HIV-RNA viral loads (VL) below quantification levels of current assays. Reasons for UL-VL detection and its relation to virological rebound (VR) are unclear. Methods: HIV-1-infected, ART-naïve patients followed at two university hospitals were included. HIV-RNA was >200 copies/mL at ART-initiation and VL<50 copies/mL achieved during ART. UL-VL was determined by the presence/absence of PCR signal detected using a commercially-available assay (COBAS® TaqMan, Roche). Random-effect Poisson regression was used for assessing determinants of UL-VL not-detected overtime and conditional risk-set analysis for VR (one VL>200 copies/mL or two VL>50 copies/mL), while accounting for frequency of VL measurements. Results: Between 2009-2013, 717 patients initiated ART containing 2 nucleos(-t)ide reverse transcriptase inhibitors (NRTI) plus a non-NRTI (29.4%), protease inhibitor (58.4%) or integrase- strand transfer inhibitor (INSTI) (12.1%). During a median 3.4 years (IQR=2.3-4.6), 676 (94.3%) patients achieved UL-VL not-detected. In multivariable analysis, UL-VL not-detected overtime was associated with younger age (p<0.001), female gender (p=0.04), lower baseline VL (p<0.001), baseline CD4+ >500 versus <350/mm (p<0.001), and INSTI-containing ART (p=0.009). 131 (18.3%) patients had VR during follow-up, which was independently associated with CD4/CD8 ratio <0.8 during follow-up (p=0.01) and time spent with UL-VL not-detected (p<0.001). When UL-VL not-detected occurred ≥50% of follow-up duration (n=290), faster time to reach UL-VL not- detected (p<0.001), faster CD4+ T-cell count increase (p=0.03), and faster CD4/CD8 ratio increase (p=0.001) were observed. Conclusions: VL-suppression at ultralow level is associated with INSTI-class ART initiation. Extensive VL-suppression below ultralow detection could improve immune reconstitution. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 INTRODUCTION Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads (VL) below levels of quantification in the majority of patients. Ultra-sensitive assays, with detection thresholds as low as 0.3 copy/mL, reveal the presence of residual viremia in treated patients [1]. The source of persistent ultra-low (UL) level viremia remains unclear, while research suggests that it could be the result of ongoing rounds of viral replication, virus released from induced latently- infected cells, or both [2]. HIV-RNA levels are associated with immunological and clinical outcomes in HIV-infected patients and hence its quantification in plasma is an essential tool for monitoring ART efficacy. Achieving and maintaining VL “undetectability” (<20 or 50 copies/mL) is the recommended target for combined- ART in all international guidelines [3,4]. For first-line regimens, patients with lower baseline HIV-RNA VL, increased genotypic susceptibility, and ART containing an integrase-strand transfer inhibitor (INSTI) or efavirenz (EFV) are more likely to exhibit virological success and less likely to encounter virological rebound (VR), defined as thresholds above 50 copies/mL [5,6]. No evidence has supported the role of HIV subtypes in virological control [7]. Since most commercially-available assays have HIV-RNA quantification thresholds at 20-50 copies/mL, it remains to be determined if these factors, and possibly others, are associated with rates of achieving HIV-RNA levels below ultra sensitive thresholds [8,9]. Furthermore, previous epidemiological studies have suggested that HIV-RNA replication detected at lower thresholds (20-40 copies/mL) increases risk of VR [10], yet whether this pertains to detection at UL-VL levels is unclear. Finally, the time-varying nature of UL-VL detectability during effective treatment has not been appropriately assessed in previous studies. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 The purpose of this study was to determine the virological, immunological and therapeutic correlates of VL below ultrasensitive detection levels (UL-VL) in a large group of ART-naïve patients from outpatient clinics initiating more recent ART combinations. Furthermore, we aimed to use long-term repeated sampling of UL-VLs during treatment in order to more concretely establish the effect of persistent residual viremia on VR. METHODS Study design and population Between 2009-2013, HIV-1-infected antiretroviral-naïve patients were selected from two French university hospitals (Pitié-Salpêtrière and Saint-Antoine Hospitals). Inclusion criteria were as follows: initiating triple ART regimen with a nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) backbone, HIV-RNA >200 copies/ml at ART-initiation, achieved <50 copies/mL at least once during ART, and ≥2 available UL-VL during follow-up. The study was carried out in accordance with the Declaration of Helsinki. Since this was a retrospective, non-interventional study with no additional procedures from standard of care and use of biological samples was permitted after receiving ethical approval (La Pitié-Salpêtrière and Saint-Antoine Hospitals, Paris, France), this study was exempt from informed consent according to French Public Health Code (CSP Article L.1121-1.1). Quantification of HIV viral load Plasma HIV-1 RNA was measured using a commercially-available PCR assay (Ampliprep COBAS TaqMan V2.0, Roche, Meylan, France). This assay provides quantitative results for HIV-RNA values Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 ≥20 copies/mL. Qualitative results are also given as HIV-RNA detected (but <20 copies/mL) or when PCR target is not detected (UL-VL not detected). Clinical and therapeutic characteristics The following patient characteristics were obtained from computerized medical records: age, gender, date of first HIV-positive serology, AIDS-defining illness, HIV subtype, CD4+ and CD8+ T- cell counts, and nadir CD4+ T-cell count. CD4/CD8 ratio was dichotomized at 0.8 (median level one-year after ART-initiation). Individual antiretroviral agents were recorded along with their dates of initiation and discontinuation, if applicable. All patients gave written informed consent that a de- identified, electronic version of their medical chart could be used for research purposes. Statistical analysis Analysis intended to reflect “real life” therapeutic options and hence follow-up incorporated patients undergoing sequential lines of combined-ART. Baseline was defined as the visit prior to ART-initiation. Patients were followed while on continuous ART until ART discontinuation, last follow-up visit, or no follow-up within 12-months after last VL measurement; whichever occurred first. Follow-up was not censored if individuals switched to another therapeutic line of ART. Two primary outcomes (based exclusively on HIV RNA levels) were analyzed in the study: (1) rate of UL-VL not detected and (2) time until VR (defined as having two consecutive HIV-RNA VL measurements >50 copies/mL or one measurement >200 copies/mL after a period of undetectability). First, in a risk-factor analysis examining rate of UL-VL not detected, univariable incident rate ratios (IRR) comparing levels of determinants and their 95% confidence intervals (CIs) were calculated using a random-effect Poisson regression model accounting for patient variability at baseline. Second, risk-factors of VR were examined in the cohort. Since VR can occur Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 several times during the course of ART, we modeled rates of VR in a repeat-event analysis [11]. Follow-up was divided in intervals of virological suppression. To summarize, the initial follow-up period began at the first visit with HIV-RNA <50 copies/mL and ended at first VR. Additional periods of follow-up were incorporated, beginning at the visit when HIV<50 copies/mL was next achieved after VR and ending at the visit with subsequent VR, until the last VR. For individuals without VR, follow-up continued until the last visit. A conditional risk-set, Cox proportional model with gap-time was used to assess determinants of VR, from which univariable hazard ratios (HR) and their 95% confidence intervals (CIs) were calculated. All parameter estimates were adjusted for number of tests per year to account for HIV-RNA testing frequency. For both the random-effect Poisson regression and conditional risk-set proportional hazards models, determinants with a p≤0.1 in univariable analysis were retained and backwards-selection was used to create a final multivariable model. Individual ARV classes were compared to all classes combined as a reference group, which is denoted as the grand mean. Several secondary outcomes were used to describe other measures of UL-VL detection over time. First, we examined the cumulative proportion achieving first UL-VL not detected. Second, we calculated the duration of follow-up with UL-VL not detected by summing, within individuals, the time intervals beginning at UL-VL not detected and ending at the next detectable UL-VL. The highest interval was taken for each individual and median (IQR) time calculated. Third, the duration of follow-up with UL-VL not detected was divided by total follow-up time for each individual to estimate overall proportion of follow-up with UL-VL not detected. We assessed the effect of changing VL levels to <20 and <50 copies/mL for all three outcomes. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 In secondary analysis, we assessed the association between proportion of follow-up time with UL- VL not detected on virological and immunological progression during treatment. Based on the upper tertile, we constructed two groups of “high” or “low” UL HIV-RNA suppression (defined at ≥50% or <50% of follow-up with UL-VL not detected, respectively). We then compared UL HIV-RNA groups with respect to time to achieving first UL-VL not detected (while testing for differences in hazards using proportional hazards regression adjusted for baseline HIV-RNA VL) and changes in CD4+ cell count and CD4/CD8 ratio from baseline (modeled as a square-root function of time using mixed- effect linear regression with random intercept and tested as a time-group interaction). All analyses were performed using STATA (v12.1, College Station, TX, USA). All statistical tests were two-sided and a p-value of <0.05 was considered significant. RESULTS Description of the study population Between 2009-2013, 717 patients initiating ART were included (519 from Pitié-Salpêtrière Hospital and 198 from Saint-Antoine Hospital). Characteristics of the study population are presented in Table 1. Most patients were male (73.5%) with median age of 39 years (IQR=32-46). Patients initiated ART shortly after HIV diagnosis (median 0.2 years, IQR=0.1-2.4). Prior to ART-initiation, median CD4 T-cell counts were not severely low, at 306/mm (IQR=178-441), and only 14.4% of patients had an AIDS-defining illness. Median baseline HIV-1 VL was 4.84 log copies/mL and 40.7% of patients presented with a VL >5.00 log copies/mL. First-line ART contained 2 NRTIs plus either an NNRTI (29.4%), protease inhibitor (PI, 58.4%) or INSTI (12.1%) (antiretroviral agents detailed in Supplementary Table 1). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Study characteristics at ART-initiation are also compared between initial ART-regimens in Table 1. A significantly higher proportion of females commenced PI-containing ART, while median age was higher in individuals commencing INSTI-containing ART. Most differences in HIV-related parameters were observed in individuals initiating PI-containing ART, with significantly higher proportion having an AIDS-defining illness, lower median current and nadir CD4+ T cell count, and higher median HIV-RNA viral load. Antiretroviral therapy and virological response during follow-up Patients were followed for a median 3.4 years (IQR=2.3-4.6). First-line therapy was continued until the end of follow-up in 292 (40.7%) patients. The remaining 425 (59.3%) patients had 1 (n=287), 2 (n=78), or ≥3 (n=60) treatment switches during follow-up, while median time until first treatment switch was 1.6 years (IQR=0.8-2.6). Among them, there were 670 switches to the following ART combinations: 2 NRTIs + 1 NNRTI (n=264), 2 NRTIs + 1 PI (n=194), 2 NRTIs + 1 INSTI (n=122), other combination ART with an NRTI backbone (n=41), dual therapy with 2 NRTIs (n=16) or with INSTI + another agent (n=11), or PI monotherapy (n=22). Overall, the median follow-up time (IQR) under the following backbones was as follows: 2 NRTIs+ 1 NNRTI, 2.0 (1.2- 3.3) years; 2 NRTIs+ 1 PI, 2.2 (1.3-3.6) years; 2 NRTIs+ 1 INSTI, 1.6 (0.7-3.1) years; and other, 1.6 (0.6-3.0) years. Individuals initiating PI-containing ART were more likely to switch regimens; however, there was no difference in the number of switches between ART regimens (Table 1). Furthermore, individuals more likely to switch ART regimens (Supplementary Table 2) were those with an AIDS-defining illness (p<0.001), lower median current and nadir CD4+ T cell count (p=0.008 and p=0.02, respectively), and higher HIV-RNA viral load (p<0.001). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 A median number of 11 (IQR=8-14) HIV-RNA VL measurements per person were taken during follow-up, resulting in a median 2 (IQR=1-3) tests per year per person. VL <50 copies/mL was achieved in all 717 patients, as part of the inclusion criteria, with 85.1%, 12.4% and 2.5% occurring during first-line, second-line and third-line ART, respectively. The cumulative proportion obtaining HIV-RNA VL <50 copies/mL was 78.1% at month 12, 86.1% at month 18 and 92.8% at month 24 of follow-up (with last patient achieving <50 copies/mL at 37 months). Suppression below ultrasensitive detection thresholds during follow-up The cumulative proportion obtaining UL-VL not detected was 42.3%, 57.7% and 70.3% at month 12, 18 and 24 of follow-up respectively. Of the 676 (94.3%) patients achieving UL-VL not detected, first UL-VL not detected occurred a median 1.1 years (IQR=0.6-2.0) after ART-initiation. Duration of UL- VL not detected lasted at most a median 1.4 years (IQR=0.7-2.3). The median proportion of follow- up with UL-VL not detected within patients was 40.0% (IQR=20.0%-60.0%), while this proportion was lower when assessed at <20 copies/mL (61.5%, IQR=47.1%-75.0%) or <50 copies/mL (71.4%, IQR=60.0%-80.0%). Clinical, immunological and virological factors associated with having an UL-VL not detected during the course of follow-up are shown in Table 2. In multivariable analysis, UL-VL not detected over time was associated with younger age (p<0.001), female gender (p=0.04), lower baseline VL (p<0.001), baseline CD4+ >500 versus <350/mm (p<0.001), and ART containing an INSTI (p=0.009). Analysis restricted to first-line therapy among individuals with virological response to their first-line regimen gave similar results (Supplementary Table 3). Patients with “high” UL HIV-RNA suppression (UL-VL not detected occurring ≥50% of the total follow-up duration) (n=290) had a significantly shorter time until achieving UL-VL not detected Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 (Figure 1A, p<0.001), faster increase in CD4+ T-cell count (Figure 1B, p=0.03), and faster increase in CD4/CD8 ratio (Figure 1C, p=0.001) than patients with low UL HIV-RNA suppression (UL-VL not detected occurring <50% of the total follow-up duration) (n=427). Virological rebound during follow-up VR occurred 237 times during follow-up and was defined by two consecutive HIV-RNA VL >50 copies/mL for 102 (43.0%) VRs, one HIV-RNA VL >200 copies/mL for 124 (52.3%) VRs, or both criteria for 11 (4.6%) VRs. Overall, VR was observed in 131 (18.3%) patients during follow-up, of whom 68 (51.9%) exhibited VR once, 41 (31.3%) twice and 22 (16.8%) three or more times. Median time until VR was 1.6 years (IQR=0.9-2.2), while only considering the first VR for those with multiple rebounds. In multivariable analysis accounting for conditional risk-sets (Table 3, Model 1), VR was associated with having a CD4/CD8 <0.8 during follow-up (p=0.01) and higher number of HIV-RNA VL tests/year (p=0.001). Of note, NNRTI-containing ART was associated with a significantly reduced risk of VR (p=0.003), and other combinations with a significantly higher risk of VR (p=0.02) in univariable analysis, yet this was not the case in multivariable analysis. When replacing cumulative duration with UL-VL not detected with ART regimen in the final multivariable model (given the collinearity between these two variables), longer periods of UL-VL suppression were significantly and inversely associated with VR (Table 3, Model 2). Analysis restricted to first-line therapy among individuals with virological response to their first-line regimen again gave similar results (Supplementary Table 4). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 DISCUSSION With advances in ART capable of providing more potent, convenient and tolerable regimens, virological suppression can be achieved in most HIV-infected patients regardless of initial first-line regimen. The main focus has now shifted towards maintaining long-term virological response and measuring the level at which suppression has been achieved. This longitudinal analysis, conducted in 717 treatment-naïve patients starting combined ART who were able to achieve virological response, demonstrated the effect of ART regimen and immune status not only on residual viremia but also subsequent VR during a median follow-up of more than 3 years. Indeed, some of the determinants more commonly associated with HIV-RNA VL suppression at higher detection thresholds were found with respect to UL-VL not detected: younger age, female gender, lower baseline VL, and higher baseline CD4+ cell count. Lower baseline plasma VLs before ART initiation and faster time to plasma virologic suppression during ART have already been shown in females compared to males [12]. The association between pre-ART VL with UL-VL not detected during treatment has also been previously highlighted in several short-term studies [8,9,13–17], suggesting a link between extensive viral spread with higher levels of circulating virus prior to ART- initiation and the infection of long-lived reservoirs [1]. One of the more important findings of our study was that ART containing an INSTI was associated with an UL-VL not detected during the course of follow-up. Our results mirror those of Gianotti et al, over a shorter time period, in which a summary statistic of percent time with residual viremia was lower among patients undergoing treatment with an INSTI [13]; yet patient populations and definition of low-level viremia were divergent from our studies. Other studies have demonstrated that HIV-RNA plasma concentrations decrease faster in the first weeks after initiating treatment Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 with INSTI-containing ART compared to PI class antiretrovirals [6,18–20] or EFV [21]. As the vast majority of patients were receiving RAL, further investigation on other INSTI agents, such as DTG, are needed. In contrast to several previous studies [13,14,22,23], no effect of NNRTI on residual viremia emerged in our study. This discrepancy could be explained by differences in NNRTI agents, while patients in our cohort were more commonly treated with rilpivirine (n=216) and efavirenz (n=188) and less so with nevirapine (n=4). Based on ultralow virological suppression, our findings tend to support recommendations of current treatment guidelines to start combined ART with an INSTI-based regimen, independent of CD4+ count and HIV-RNA VL [3,4]. Since persistent residual viremia appears to impact immune activation, inflammation and microbial translocation [24,25], VLs at ultralow levels could bear some clinical importance. The ongoing presence of virus, despite low viral concentrations, could prevent some systemic inflammatory markers from returning to normal levels; however, the association between residual inflammation and residual HIV-1 replication is probably bi-directional and complex [26]. Indeed, as shown in several studies among patients undergoing treatment intensification [27–31] or effective ART for seven years [27], residual viremia under ART has been associated with low-grade immune activation. Another study has shown that immune activation still persists despite UL-VL not detected after 10 years of treatment [28], but was assessed with only one cross-sectional measure of UL-VL. Several studies have shown that restoration of the CD4/CD8 ratio is associated with faster virological control after ART initiation (VL <50 copies/mL) [29,30]. At the same time, CD4 decline appears to be accelerated in studies investigating immunologic progression among elite HIV controllers without ART [29–31]. In our study, we observed that patients with more extensive viral suppression below ultrasensitive detection levels during ART, defined as ≥50% of follow-up with Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 UL-VL not detected, have faster increases of CD4 cell count and CD4/CD8 ratio. Interestingly, CD4/CD8 ratio is inversely correlated to levels of inflammation and immune activation in patients with virological suppression [32]. Whether the immunological effects linked to increased UL-VL suppression also corresponds to improvement in inflammation remains to be fully elucidated. The relationship between HIV-RNA replication and VR has been examined in several studies using assays with higher detection thresholds, but this association with respect to residual viremia is unclear. Past research has shown that patients with slower time to achieving <50 copies/mL are more likely to continue treatment with residual viremia [13] and, when <50 copies/mL occurs after 6 months from initiating ART, have an almost 2-fold risk of subsequent VR [6]. Reports with clearer evidence state that the inability to achieve UL-VL during first-line therapy could lead to a higher risk of VR in certain settings [33,34], while others fail to observe similar results [35]. In our study, we observed that patients without VR were able to suppress UL-VL not detected at a significantly faster rate, similar to patients with “high” suppression of UL HIV-RNA during the course of treatment. Furthermore, we demonstrated that the longer UL-VL not detected occurred during treatment duration, the lower the risk of VR. These results suggest the importance of extensive long-term suppression in order to avoid subsequent VR. Our study has several noteworthy advantages. The large number of patients with many repeated samples, including HIV-RNA VL measures, is one of the most extensive to date. These data allow more flexibility in analytical options, hence why we decided to examine not only proportion of viral suppression during follow-up, as have others [13], but more appropriately, the risk factors associated with the rate of ultralow viral suppression across all visits over time. This is an important distinction as proportion of viral suppression can be biased by length of follow-up and most studies have evaluated this statistic at a single time-point (i.e. last follow-up visit). Furthermore, we did not Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 focus only on first-line ART regimens or time until first VR, but rather the overall therapeutic strategies adapted throughout follow-up (by including time-varying treatment switches in the models) and the multiple rounds of VR (via conditional risk-set proportional hazards models). Including this information provides a more accurate representation of the dynamic processes occurring during routine follow-up. Despite these advantages, our study has some limitations. First, this study lacks information on adherence, pharmacological data and ART-associated adverse events, which could play a role in UL- VL detection [36,37]. Second, we did not analyze data on genotypic resistance at the start of treatment or at the time of rebound. Third, HIV-RNA VLs were measured using a commercially- available assay as part of clinical follow up. Single Copy Assays are more adapted to measuring ultrasensitive detection; nevertheless, this time- and cost-prohibitive assay requires large amounts of blood to be collected and cannot be readily used in routine practice [34]. The major limitation of routine PCR is that when HIV RNA VLs are extremely low, within-assay variation is large and could have an impact on reproducibility when evaluating detection or within-patient variation [38]. It should be mentioned, however, that other studies investigating residual viremia have also used real- time PCR assays with fairly reproducible conclusions [10,13,17,33]. Finally, although estimates were adjusted for the number of tests per year, we cannot completely exclude missed virological rebounds. Finally, PI-containing regimens were more often given to individuals with high HIV-RNA VL and low CD4+ T-cell count at ART-initiation and this bias could have influenced our results. Although we did adjust for these factors in multivariable analysis, residual confounding cannot be fully excluded. In conclusion, notwithstanding the non-randomized allocation of treatment combinations, we showed that diverse ART regimens have different effects on residual viremia after achieving <50 Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 HIV-RNA copies/mL. In particular, higher rates of virological suppression with UL-VL not detected are more common in individuals undergoing INSTI-based ART regimens. Moreover, given the association between residual viremia and immune activation, INSTI class antiretrovirals, a preferred third agent for first-line ART according to current recommendations, could also lead to improvement in immunological parameters and possibly reduced inflammation. Further evidence would be needed, however, on other INSTI class agents to confirm this hypothesis. FUNDING This work was supported by the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales. TRANSPARENCY DECLARATIONS S.L has received travel grants from Gilead Sciences and ViiV Healthcare. JLM has received travel grants from Abbvie Labs, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare and has received honoraria for invited talks from Gilead Sciences, Merck Laboratories MSD and ViiV Healthcare. PMG has received grant support from BMS and Janssen Pharmaceuticals, personal fees as member of symposia faculty from BMS, Abbvie Labs, and Mylan and honoraria for participation on international boards from Gilead Sciences and ViiV Healthcare. CK has received grant support from Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. A. G. M. has received honoraria for advisories or invited talks or conferences and research grants from Abbvie Labs, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. LMJ have received honoraria from BMS, Mylan, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. The other authors have none to declare. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 REFERENCES 1. Palmer S, Maldarelli F, Wiegand A, et al. 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Available at: http://content.wkhealth.com/linkback/openurl?sid=WKPTLP:landingpage&an=00002030- 201101280-00008. Accessed 29 December 2015. 24. Boyd A, Meynard J-L, Morand-Joubert L, et al. Association of Residual Plasma Viremia and Intima-Media Thickness in Antiretroviral-Treated Patients with Controlled Human Immunodeficiency Virus Infection. PLoS ONE 2014; 9. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240670/. Accessed 30 December 2015. 25. Bastard J-P, Soulié C, Fellahi S, et al. Circulating interleukin-6 levels correlate with residual HIV viraemia and markers of immune dysfunction in treatment-controlled HIV-infected patients. Antivir Ther 2012; 17:915–919. 26. Martinez-Picado J, Deeks SG. Persistent HIV-1 replication during antiretroviral therapy. Curr Opin HIV AIDS 2016; 11:417–423. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 27. Mavigner M, Delobel P, Cazabat M, et al. HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy. PLoS ONE 2009; 4. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765414/. Accessed 29 December 28. Guihot A, Dentone C, Assoumou L, et al. Residual immune activation in combined antiretroviral therapy- treated patients with maximally suppressed viremia. AIDS Lond Engl 2016; 30:327–330. 29. Noel N, Lerolle N, Lécuroux C, et al. Immunologic and Virologic Progression in HIV Controllers: The Role of Viral ‘Blips’ and Immune Activation in the ANRS CO21 CODEX Study. PloS One 2015; 10:e0131922. 30. Boufassa F, Saez-Cirion A, Lechenadec J, et al. CD4 dynamics over a 15 year-period among HIV controllers enrolled in the ANRS French observatory. PloS One 2011; 6:e18726. 31. Pereyra F, Palmer S, Miura T, et al. Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters. J Infect Dis 2009; 200:984–990. 32. Zheng L, Taiwo B, Gandhi RT, et al. Factors associated with CD8+ T-cell activation in HIV-1-infected patients on long-term antiretroviral therapy. J Acquir Immune Defic Syndr 1999 2014; 67:153–160. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167746/. Accessed 31 December 2015. 33. Gianotti N, Galli L, Salpietro S, et al. Virological rebound in human immunodeficiency virus-infected patients with or without residual viraemia: results from an extended follow-up. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2013; 19:E542-544. 34. Calcagno A, Motta I, Ghisetti V, et al. HIV-1 Very Low Level Viremia Is Associated with Virological Failure in Highly Active Antiretroviral Treatment-Treated Patients. AIDS Res Hum Retroviruses 2015; 31:999– 35. Helou E, Shenoi S, Kyriakides T, Landry M-L, Kozal M, Barakat LA. Characterizing Patients with Very- Low-Level HIV Viremia: A Community-Based Study. J Int Assoc Provid AIDS Care 2017; 16:261–266. 36. Leierer G, Grabmeier-Pfistershammer K, Steuer A, et al. Factors Associated with Low-Level Viraemia and Virological Failure: Results from the Austrian HIV Cohort Study. PloS One 2015; 10:e0142923. 37. Pasternak AO, de Bruin M, Jurriaans S, et al. Modest nonadherence to antiretroviral therapy promotes residual HIV-1 replication in the absence of virological rebound in plasma. J Infect Dis 2012; 206:1443– 38. Ruelle J, Debaisieux L, Vancutsem E, et al. HIV-1 low-level viraemia assessed with 3 commercial real- time PCR assays show high variability. BMC Infect Dis 2012; 12:100. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 TABLES Table 1. Description of the study population at antiretroviral therapy initiation Initial ART backbone NRTI+NNRTI NRTI+PI NRTI+INSTI N=717 (n=211) (n=419) (n=87) p Male/Female (% males) 527/190 (73.5) 171/40 (81.0) 293/126 (69.9) 63/24 (72.4) 0.01 Age* [N=716] 39 (32-46) 39 (32-47) 38 (32-45) 42 (34-51) 0.009 Hospital 0.005 La Pitié-Salpétrière 519 (72.4) 160 (75.8) 286 (68.3) 73 (83.9) Saint-Antoine 198 (27.6) 51 (24.2) 133 (31.7) 14 (16.1) Time since first positive HIV serology*, years 0.2 (0.1-2.4) 0.5 (0.1-2.5) 0.2 (0.1-2.3) 0.1 (0.1-2.9) 0.2 AIDS-defining illness 103 (14.4) 16 (7.8) 75 (17.9) 12 (13.8) 0.002 CD4+ T-cell count*, /mm [N=666] 306 (178-441) 366 (268-467) 275 (137-408) 322 (210-408) <0.001 Nadir CD4+ T-cell count*, mm 275 (157-374) 328 (242-410) 238 (110-349) 278 (168-360) <0.001 CD8+ T-cell count*, /mm [N=643] 780 (538-1141) 822 (586-1241) 764 (497-1115) 780 (564-1167) 0.10 CD4:CD8 ratio* [N=643] 0.35 (0.21-0.57) 0.41 (0.24-0.63) 0.31 (0.17-0.51) 0.35 (0.21-0.57) <0.001 HIV-RNA viral load*, log copies/mL 4.84 (4.36-5.24) 4.68 (4.17-5.08) 4.90 (4.41-5.33) 4.84 (4.49-5.29) <0.001 HIV-RNA viral load >10 copies/mL 292 (40.7) 66 (31.3) 187 (44.6) 39 (44.8) 0.004 HIV subtype [N=105] 0.7 B 43 (41.0) 12 (48.0) 25 (37.3) 6 (46.2) CRF02_AG 34 (32.4) 6 (24.0) 25 (37.3) 3 (23.1) Other 28 (26.7) 7 (28.0) 17 (25.4) 4 (30.8) Number of antiretroviral agents* 3 (3-3) 3 (3-3) 3 (3-3) 3 (3-3) 0.6 Positive HCV RNA [N=291] 18 (6.2) 4 (5.5) 7 (3.9) 7 (18.9) 0.002 HCV RNA viral load, log IU/mL* 6.20 (5.70-6.55) 6.20 (5.52-6.56) 6.55 (5.88-6.71) 6.15 (5.63-6.39) 0.5 Positive HBsAg serology [N=705] 23 (3.3) 5 (2.4) 18 (4.3) 0 (0) 0.09 Switching ART regiment during follow-up 425 (59.3) 92 (43.6) 284 (67.8) 49 (56.3) <0.001 Number of switches during follow-up* 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 0.8 All statistics are n (%), except for continuous variables (*) where medians (IQR) are reported. † ‡ Only among patients with positive HCV RNA viral loads. Only among patients switching ART regiments at least once. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Table 2. Determinants of UL-VL not detected during antiretroviral therapy Univariable Multivariable* IRR (95%CI) p IRR (95%CI) p Age (per 10 years) [N=716] 0.88 (0.83-0.92) <0.001 0.89 (0.85-0.94) <0.001 Gender Male 1.00 1.00 Female 1.13 (1.00-1.28) 0.06 1.14 (1.01-1.29) 0.04 Baseline HIV-RNA <10 copies/mL 1.00 1.00 ≥10 copies/mL 0.67 (0.60-0.75) <0.001 0.72 (0.64-0.80) <0.001 Baseline CD4+ T-cell count [N=665] >500 cells/mm 1.00 1.00 350-500 cells/mm 0.91 (0.77-1.08) 0.3 0.90 (0.76-1.06) 0.2 <350 cells/mm 0.63 (0.54-0.74) <0.001 0.66 (0.57-0.77) <0.001 Baseline CD4+ T-cell nadir ≥250 cells/mm 1.00 <250 cells/ mm 0.68 (0.61-0.76) <0.001 Time-varying CD4:CD8 ratio [N=681] ≥0.8 1.00 <0.8 0.89 (0.81-0.99) 0.03 Anchor drug of ART during follow-up NNRTI 0.99 (0.91-1.07) 0.8 0.99 (0.91-1.07) 0.8 PI 0.98 (0.91-1.07) 0.7 0.96 (0.89-1.05) 0.4 Integrase inhibitor 1.13 (1.01-1.25) 0.03 1.15 (1.04-1.28) 0.009 Other combination 0.91 (0.79-1.06) 0.2 0.91 (0.78-1.06) 0.2 Concomitant treatment switch No 1.00 Yes 0.97 (0.86-1.09) 0.6 Analysis includes 717 HIV-infected patients initiating antiretroviral therapy (ART) and accounts for subsequent lines of ART after switching regimens. Incident rate ratios (IRR) and 95% confidence intervals (CIs) calculated from a random-effect Poisson regression accounting for patient variability at baseline. *In multivariable analysis, baseline nadir CD4+ was closely linked to baseline CD4+ T-cell count and hence excluded. Time-varying CD4:CD8 ratio (preferred over CD4:CD8 ratio at baseline) was excluded because its associated p-value was below the pre-specified threshold (p=0.402). A total of 52 patients had missing data on baseline CD4 T-cell count (n=51) or age (n=1) and were not included the multivariable model. Individual ARV classes are compared to all classes combined as a reference group. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Table 3. Determinants of virological rebound after antiretroviral therapy initiation Multivariable** Multivariable*** Univariable* Model 1 Model 2 HR (95%CI) p HR (95%CI) p HR (95%CI) p 0.95 (0.80- Age (per 10 years) [N=716] 1.14) 0.6 Gender Male 1.00 0.72 (0.46- Female 1.11) 0.14 Baseline HIV-RNA <10 copies/mL 1.00 1.00 1.00 1.43 (0.99- 1.45 (0.96- 1.18 (0.78- ≥10 copies/mL 2.09) 0.06 2.19) 0.08 1.79) 0.4 Cumulative duration with UL-VL 0.41 (0.29- 0.45 (0.30- not detected 0.58) <0.001 0.67) <0.001 Baseline CD4+ T-cell count [N=666] >500 cells/mm 1.00 1.51 (0.83- 350-500 cells/mm 2.74) 0.18 1.25 (0.71- <350 cells/mm 2.19) 0.4 Baseline CD4+ T-cell nadir ≥250 cells/mm 1.00 0.86 (0.57- <250 cells/mm 1.30) 0.5 Time-varying CD4:CD8 ratio [N=681] ≥0.8 1.00 1.00 1.00 2.04 (1.32- 1.95 (1.17- 1.87 (1.15- <0.8 3.18) 0.001 3.24) 0.01 3.05) 0.01 Anchor drug of ART during follow- up 0.60 (0.43- 0.76 (0.50- NNRTI 0.84) 0.003 1.15) 0.2 - 1.20 (0.88- 1.00 (0.69- PI 1.64) 0.2 1.43) 0.9 - 0.82 (0.48- 1.23 (0.64- Integrase inhibitor 1.40) 0.5 2.33) 0.5 - 1.68 (1.11- 1.08 (0.66- Other combination 2.56) 0.02 1.75) 0.8 - Concomitant treatment switch No 1.00 1.48 (0.94- Yes 2.32) 0.09 Number of HIV-RNA tests per year ≤3 0.33 (0.18- <0.001 0.29 (0.14- 0.002 0.24 (0.12- <0.001 Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 0.62) 0.63) 0.49) 0.78 (0.39- 0.83 (0.36- 0.66 (0.31- 3-4 1.58) 0.5 1.93) 0.7 1.40) 0.3 1.25 (0.63- 1.29 (0.61- 0.99 (0.50- 4-6 2.50) 0.5 2.71) 0.5 1.95) 0.9 >6 1.00 1.00 1.00 Analysis includes 717 HIV-infected patients initiating antiretroviral therapy (ART) and accounts for subsequent lines of ART after switching regimens. Hazard ratios (HR) and 95% confidence intervals (CIs) were estimated from a conditional risk-set, Cox proportional hazards model (using time from the previous event). *Adjusted for number of HIV-RNA tests per year to account for individuals with more frequent testing (hence, higher possibility of detecting VR). **In the multivariable model, treatment switch was excluded from the model as its associated p-value was below the pre-specified threshold (p=0.157). A total of 36 patients had missing data on CD4:CD8 ratio and were not included in the multivariable model. ***Replacing cumulative duration with UL-VL not detected as a time-varying variable with ART regimen in Model 1 (given the collinearity between these two variables). Individual ARV classes are compared to all classes combined as a reference group. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 FIGURE LEGENDS Figure 1. Time to reach UL-VL not detected and changes in immunological parameters after initiating antiretroviral therapy according to residual viremia and virological rebound Cumulative proportion of patients reaching an undetectable UL-VL with detection threshold at ultrasensitive levels is depicted in (A), while adjusted for baseline HIV-RNA viral load. Locally- weighted scatterplot smoothing lines are constructed for CD4+ T-cell count (B) and CD4+:CD8+ ratio (C) during follow-up. All figures are stratified on patients with low versus high HIV-RNA suppression (defined as <50% versus ≥50%, respectively, of follow-up spent with an UL-VL not detected). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Figure 1. 1.00 0.80 p<0.001 0.60 0.40 0.20 L ow US suppr High US suppr 0.00 0 1 2 3 4 5 6 T ime (years ) p=0.03 L ow US s uppr High US s uppr 0 1 2 3 4 T ime (years ) 1.2 1.0 0.8 p=0.001 0.6 0.4 0.2 L ow US suppr High US suppr 0.0 0 1 2 3 4 T ime (years ) Accepted Manuscript C umulative probability C D4+ cell count (/mm3) C D4+:C D8+ ratio http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

INSTI-based triple regimens in treatment-naïve HIV-infected patients are associated with HIV-RNA viral load suppression at ultralow levels

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Oxford University Press
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© The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
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10.1093/ofid/ofz177
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Abstract

Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 INSTI-based triple regimens in treatment-naïve HIV-infected patients are associated with HIV-RNA viral load suppression at ultralow levels 1, 2, 3 2 2, 3 4 2, 5 Sidonie Lambert-Niclot , Anders Boyd , Djeneba Fofana , Nadia Valin , Marc Wirden , Jean-Luc 4 1, 2, 6 6 1, 2, 6 1, 2, 5 Meynard , Romain Palich , Rachid Agher , Marc-Antoine Valantin , Vincent Calvez , 1, 2, 6 1, 2, 4 1, 2, 5 Christine Katlama , Pierre-Marie Girard , Anne-Geneviève Marcelin , Laurence Morand- 1, 2, 3 Joubert Institutional affiliations: Sorbonne Université, Paris France INSERM, Institut Pierre Louis d’Epidémiologie et de Santé Publique (iPLESP), F-75012 Paris, France AP-HP, Hôpital Saint-Antoine, Laboratoire de virologie, F-75012 Paris, France AP-HP, Hôpital Saint-Antoine, Département de Maladies Infectieuses, F75012, Paris, France; AP-HP, Hôpital Pitié Salpêtrière, Laboratoire de virologie, F-75013 Paris, France ; AP-HP, Hôpital Pitié Salpêtrière, Département de Maladies Infectieuses, F75013, Paris, France; Keywords: HIV, initiation antiretroviral therapy, integrase inhibitor, residual viremia. Keypoints: In treated HIV-1-infected patients, viral load-suppression at ultralow levels is associated with INSTI-class ART. More time spent with UL-VL undetectable appears to improve immunoreconstitution and, along with CD4/CD8 ratio >0.8 over time, reduce the risk of virological rebound. © The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non- commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Corresponding author: Sidonie Lambert-Niclot, PharmD, PhD Department of Virology, Saint-Antoine Hospital 184 rue du faubourg Saint-Antoine 75571 Paris Cedex 12 Tel: +33 1 71 97 09 51 Fax: +33 1 49 28 24 82 E-mail: sidonie.lambert@aphp.fr Alternative corresponding author: Laurence Morand-Joubert,MD, PhD Department of Virology, Saint-Antoine Hospital 184 rue du faubourg Saint-Antoine 75571 Paris Cedex 12 Tel: +33 1 71 97 09 51 Fax: +33 1 49 28 24 82 E-mail: laurence.morand-joubert@aphp.fr Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 ABSTRACT Background: During antiretroviral therapy (ART), HIV-1-infected patients may present with ultralow (UL) HIV-RNA viral loads (VL) below quantification levels of current assays. Reasons for UL-VL detection and its relation to virological rebound (VR) are unclear. Methods: HIV-1-infected, ART-naïve patients followed at two university hospitals were included. HIV-RNA was >200 copies/mL at ART-initiation and VL<50 copies/mL achieved during ART. UL-VL was determined by the presence/absence of PCR signal detected using a commercially-available assay (COBAS® TaqMan, Roche). Random-effect Poisson regression was used for assessing determinants of UL-VL not-detected overtime and conditional risk-set analysis for VR (one VL>200 copies/mL or two VL>50 copies/mL), while accounting for frequency of VL measurements. Results: Between 2009-2013, 717 patients initiated ART containing 2 nucleos(-t)ide reverse transcriptase inhibitors (NRTI) plus a non-NRTI (29.4%), protease inhibitor (58.4%) or integrase- strand transfer inhibitor (INSTI) (12.1%). During a median 3.4 years (IQR=2.3-4.6), 676 (94.3%) patients achieved UL-VL not-detected. In multivariable analysis, UL-VL not-detected overtime was associated with younger age (p<0.001), female gender (p=0.04), lower baseline VL (p<0.001), baseline CD4+ >500 versus <350/mm (p<0.001), and INSTI-containing ART (p=0.009). 131 (18.3%) patients had VR during follow-up, which was independently associated with CD4/CD8 ratio <0.8 during follow-up (p=0.01) and time spent with UL-VL not-detected (p<0.001). When UL-VL not-detected occurred ≥50% of follow-up duration (n=290), faster time to reach UL-VL not- detected (p<0.001), faster CD4+ T-cell count increase (p=0.03), and faster CD4/CD8 ratio increase (p=0.001) were observed. Conclusions: VL-suppression at ultralow level is associated with INSTI-class ART initiation. Extensive VL-suppression below ultralow detection could improve immune reconstitution. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 INTRODUCTION Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads (VL) below levels of quantification in the majority of patients. Ultra-sensitive assays, with detection thresholds as low as 0.3 copy/mL, reveal the presence of residual viremia in treated patients [1]. The source of persistent ultra-low (UL) level viremia remains unclear, while research suggests that it could be the result of ongoing rounds of viral replication, virus released from induced latently- infected cells, or both [2]. HIV-RNA levels are associated with immunological and clinical outcomes in HIV-infected patients and hence its quantification in plasma is an essential tool for monitoring ART efficacy. Achieving and maintaining VL “undetectability” (<20 or 50 copies/mL) is the recommended target for combined- ART in all international guidelines [3,4]. For first-line regimens, patients with lower baseline HIV-RNA VL, increased genotypic susceptibility, and ART containing an integrase-strand transfer inhibitor (INSTI) or efavirenz (EFV) are more likely to exhibit virological success and less likely to encounter virological rebound (VR), defined as thresholds above 50 copies/mL [5,6]. No evidence has supported the role of HIV subtypes in virological control [7]. Since most commercially-available assays have HIV-RNA quantification thresholds at 20-50 copies/mL, it remains to be determined if these factors, and possibly others, are associated with rates of achieving HIV-RNA levels below ultra sensitive thresholds [8,9]. Furthermore, previous epidemiological studies have suggested that HIV-RNA replication detected at lower thresholds (20-40 copies/mL) increases risk of VR [10], yet whether this pertains to detection at UL-VL levels is unclear. Finally, the time-varying nature of UL-VL detectability during effective treatment has not been appropriately assessed in previous studies. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 The purpose of this study was to determine the virological, immunological and therapeutic correlates of VL below ultrasensitive detection levels (UL-VL) in a large group of ART-naïve patients from outpatient clinics initiating more recent ART combinations. Furthermore, we aimed to use long-term repeated sampling of UL-VLs during treatment in order to more concretely establish the effect of persistent residual viremia on VR. METHODS Study design and population Between 2009-2013, HIV-1-infected antiretroviral-naïve patients were selected from two French university hospitals (Pitié-Salpêtrière and Saint-Antoine Hospitals). Inclusion criteria were as follows: initiating triple ART regimen with a nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) backbone, HIV-RNA >200 copies/ml at ART-initiation, achieved <50 copies/mL at least once during ART, and ≥2 available UL-VL during follow-up. The study was carried out in accordance with the Declaration of Helsinki. Since this was a retrospective, non-interventional study with no additional procedures from standard of care and use of biological samples was permitted after receiving ethical approval (La Pitié-Salpêtrière and Saint-Antoine Hospitals, Paris, France), this study was exempt from informed consent according to French Public Health Code (CSP Article L.1121-1.1). Quantification of HIV viral load Plasma HIV-1 RNA was measured using a commercially-available PCR assay (Ampliprep COBAS TaqMan V2.0, Roche, Meylan, France). This assay provides quantitative results for HIV-RNA values Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 ≥20 copies/mL. Qualitative results are also given as HIV-RNA detected (but <20 copies/mL) or when PCR target is not detected (UL-VL not detected). Clinical and therapeutic characteristics The following patient characteristics were obtained from computerized medical records: age, gender, date of first HIV-positive serology, AIDS-defining illness, HIV subtype, CD4+ and CD8+ T- cell counts, and nadir CD4+ T-cell count. CD4/CD8 ratio was dichotomized at 0.8 (median level one-year after ART-initiation). Individual antiretroviral agents were recorded along with their dates of initiation and discontinuation, if applicable. All patients gave written informed consent that a de- identified, electronic version of their medical chart could be used for research purposes. Statistical analysis Analysis intended to reflect “real life” therapeutic options and hence follow-up incorporated patients undergoing sequential lines of combined-ART. Baseline was defined as the visit prior to ART-initiation. Patients were followed while on continuous ART until ART discontinuation, last follow-up visit, or no follow-up within 12-months after last VL measurement; whichever occurred first. Follow-up was not censored if individuals switched to another therapeutic line of ART. Two primary outcomes (based exclusively on HIV RNA levels) were analyzed in the study: (1) rate of UL-VL not detected and (2) time until VR (defined as having two consecutive HIV-RNA VL measurements >50 copies/mL or one measurement >200 copies/mL after a period of undetectability). First, in a risk-factor analysis examining rate of UL-VL not detected, univariable incident rate ratios (IRR) comparing levels of determinants and their 95% confidence intervals (CIs) were calculated using a random-effect Poisson regression model accounting for patient variability at baseline. Second, risk-factors of VR were examined in the cohort. Since VR can occur Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 several times during the course of ART, we modeled rates of VR in a repeat-event analysis [11]. Follow-up was divided in intervals of virological suppression. To summarize, the initial follow-up period began at the first visit with HIV-RNA <50 copies/mL and ended at first VR. Additional periods of follow-up were incorporated, beginning at the visit when HIV<50 copies/mL was next achieved after VR and ending at the visit with subsequent VR, until the last VR. For individuals without VR, follow-up continued until the last visit. A conditional risk-set, Cox proportional model with gap-time was used to assess determinants of VR, from which univariable hazard ratios (HR) and their 95% confidence intervals (CIs) were calculated. All parameter estimates were adjusted for number of tests per year to account for HIV-RNA testing frequency. For both the random-effect Poisson regression and conditional risk-set proportional hazards models, determinants with a p≤0.1 in univariable analysis were retained and backwards-selection was used to create a final multivariable model. Individual ARV classes were compared to all classes combined as a reference group, which is denoted as the grand mean. Several secondary outcomes were used to describe other measures of UL-VL detection over time. First, we examined the cumulative proportion achieving first UL-VL not detected. Second, we calculated the duration of follow-up with UL-VL not detected by summing, within individuals, the time intervals beginning at UL-VL not detected and ending at the next detectable UL-VL. The highest interval was taken for each individual and median (IQR) time calculated. Third, the duration of follow-up with UL-VL not detected was divided by total follow-up time for each individual to estimate overall proportion of follow-up with UL-VL not detected. We assessed the effect of changing VL levels to <20 and <50 copies/mL for all three outcomes. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 In secondary analysis, we assessed the association between proportion of follow-up time with UL- VL not detected on virological and immunological progression during treatment. Based on the upper tertile, we constructed two groups of “high” or “low” UL HIV-RNA suppression (defined at ≥50% or <50% of follow-up with UL-VL not detected, respectively). We then compared UL HIV-RNA groups with respect to time to achieving first UL-VL not detected (while testing for differences in hazards using proportional hazards regression adjusted for baseline HIV-RNA VL) and changes in CD4+ cell count and CD4/CD8 ratio from baseline (modeled as a square-root function of time using mixed- effect linear regression with random intercept and tested as a time-group interaction). All analyses were performed using STATA (v12.1, College Station, TX, USA). All statistical tests were two-sided and a p-value of <0.05 was considered significant. RESULTS Description of the study population Between 2009-2013, 717 patients initiating ART were included (519 from Pitié-Salpêtrière Hospital and 198 from Saint-Antoine Hospital). Characteristics of the study population are presented in Table 1. Most patients were male (73.5%) with median age of 39 years (IQR=32-46). Patients initiated ART shortly after HIV diagnosis (median 0.2 years, IQR=0.1-2.4). Prior to ART-initiation, median CD4 T-cell counts were not severely low, at 306/mm (IQR=178-441), and only 14.4% of patients had an AIDS-defining illness. Median baseline HIV-1 VL was 4.84 log copies/mL and 40.7% of patients presented with a VL >5.00 log copies/mL. First-line ART contained 2 NRTIs plus either an NNRTI (29.4%), protease inhibitor (PI, 58.4%) or INSTI (12.1%) (antiretroviral agents detailed in Supplementary Table 1). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Study characteristics at ART-initiation are also compared between initial ART-regimens in Table 1. A significantly higher proportion of females commenced PI-containing ART, while median age was higher in individuals commencing INSTI-containing ART. Most differences in HIV-related parameters were observed in individuals initiating PI-containing ART, with significantly higher proportion having an AIDS-defining illness, lower median current and nadir CD4+ T cell count, and higher median HIV-RNA viral load. Antiretroviral therapy and virological response during follow-up Patients were followed for a median 3.4 years (IQR=2.3-4.6). First-line therapy was continued until the end of follow-up in 292 (40.7%) patients. The remaining 425 (59.3%) patients had 1 (n=287), 2 (n=78), or ≥3 (n=60) treatment switches during follow-up, while median time until first treatment switch was 1.6 years (IQR=0.8-2.6). Among them, there were 670 switches to the following ART combinations: 2 NRTIs + 1 NNRTI (n=264), 2 NRTIs + 1 PI (n=194), 2 NRTIs + 1 INSTI (n=122), other combination ART with an NRTI backbone (n=41), dual therapy with 2 NRTIs (n=16) or with INSTI + another agent (n=11), or PI monotherapy (n=22). Overall, the median follow-up time (IQR) under the following backbones was as follows: 2 NRTIs+ 1 NNRTI, 2.0 (1.2- 3.3) years; 2 NRTIs+ 1 PI, 2.2 (1.3-3.6) years; 2 NRTIs+ 1 INSTI, 1.6 (0.7-3.1) years; and other, 1.6 (0.6-3.0) years. Individuals initiating PI-containing ART were more likely to switch regimens; however, there was no difference in the number of switches between ART regimens (Table 1). Furthermore, individuals more likely to switch ART regimens (Supplementary Table 2) were those with an AIDS-defining illness (p<0.001), lower median current and nadir CD4+ T cell count (p=0.008 and p=0.02, respectively), and higher HIV-RNA viral load (p<0.001). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 A median number of 11 (IQR=8-14) HIV-RNA VL measurements per person were taken during follow-up, resulting in a median 2 (IQR=1-3) tests per year per person. VL <50 copies/mL was achieved in all 717 patients, as part of the inclusion criteria, with 85.1%, 12.4% and 2.5% occurring during first-line, second-line and third-line ART, respectively. The cumulative proportion obtaining HIV-RNA VL <50 copies/mL was 78.1% at month 12, 86.1% at month 18 and 92.8% at month 24 of follow-up (with last patient achieving <50 copies/mL at 37 months). Suppression below ultrasensitive detection thresholds during follow-up The cumulative proportion obtaining UL-VL not detected was 42.3%, 57.7% and 70.3% at month 12, 18 and 24 of follow-up respectively. Of the 676 (94.3%) patients achieving UL-VL not detected, first UL-VL not detected occurred a median 1.1 years (IQR=0.6-2.0) after ART-initiation. Duration of UL- VL not detected lasted at most a median 1.4 years (IQR=0.7-2.3). The median proportion of follow- up with UL-VL not detected within patients was 40.0% (IQR=20.0%-60.0%), while this proportion was lower when assessed at <20 copies/mL (61.5%, IQR=47.1%-75.0%) or <50 copies/mL (71.4%, IQR=60.0%-80.0%). Clinical, immunological and virological factors associated with having an UL-VL not detected during the course of follow-up are shown in Table 2. In multivariable analysis, UL-VL not detected over time was associated with younger age (p<0.001), female gender (p=0.04), lower baseline VL (p<0.001), baseline CD4+ >500 versus <350/mm (p<0.001), and ART containing an INSTI (p=0.009). Analysis restricted to first-line therapy among individuals with virological response to their first-line regimen gave similar results (Supplementary Table 3). Patients with “high” UL HIV-RNA suppression (UL-VL not detected occurring ≥50% of the total follow-up duration) (n=290) had a significantly shorter time until achieving UL-VL not detected Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 (Figure 1A, p<0.001), faster increase in CD4+ T-cell count (Figure 1B, p=0.03), and faster increase in CD4/CD8 ratio (Figure 1C, p=0.001) than patients with low UL HIV-RNA suppression (UL-VL not detected occurring <50% of the total follow-up duration) (n=427). Virological rebound during follow-up VR occurred 237 times during follow-up and was defined by two consecutive HIV-RNA VL >50 copies/mL for 102 (43.0%) VRs, one HIV-RNA VL >200 copies/mL for 124 (52.3%) VRs, or both criteria for 11 (4.6%) VRs. Overall, VR was observed in 131 (18.3%) patients during follow-up, of whom 68 (51.9%) exhibited VR once, 41 (31.3%) twice and 22 (16.8%) three or more times. Median time until VR was 1.6 years (IQR=0.9-2.2), while only considering the first VR for those with multiple rebounds. In multivariable analysis accounting for conditional risk-sets (Table 3, Model 1), VR was associated with having a CD4/CD8 <0.8 during follow-up (p=0.01) and higher number of HIV-RNA VL tests/year (p=0.001). Of note, NNRTI-containing ART was associated with a significantly reduced risk of VR (p=0.003), and other combinations with a significantly higher risk of VR (p=0.02) in univariable analysis, yet this was not the case in multivariable analysis. When replacing cumulative duration with UL-VL not detected with ART regimen in the final multivariable model (given the collinearity between these two variables), longer periods of UL-VL suppression were significantly and inversely associated with VR (Table 3, Model 2). Analysis restricted to first-line therapy among individuals with virological response to their first-line regimen again gave similar results (Supplementary Table 4). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 DISCUSSION With advances in ART capable of providing more potent, convenient and tolerable regimens, virological suppression can be achieved in most HIV-infected patients regardless of initial first-line regimen. The main focus has now shifted towards maintaining long-term virological response and measuring the level at which suppression has been achieved. This longitudinal analysis, conducted in 717 treatment-naïve patients starting combined ART who were able to achieve virological response, demonstrated the effect of ART regimen and immune status not only on residual viremia but also subsequent VR during a median follow-up of more than 3 years. Indeed, some of the determinants more commonly associated with HIV-RNA VL suppression at higher detection thresholds were found with respect to UL-VL not detected: younger age, female gender, lower baseline VL, and higher baseline CD4+ cell count. Lower baseline plasma VLs before ART initiation and faster time to plasma virologic suppression during ART have already been shown in females compared to males [12]. The association between pre-ART VL with UL-VL not detected during treatment has also been previously highlighted in several short-term studies [8,9,13–17], suggesting a link between extensive viral spread with higher levels of circulating virus prior to ART- initiation and the infection of long-lived reservoirs [1]. One of the more important findings of our study was that ART containing an INSTI was associated with an UL-VL not detected during the course of follow-up. Our results mirror those of Gianotti et al, over a shorter time period, in which a summary statistic of percent time with residual viremia was lower among patients undergoing treatment with an INSTI [13]; yet patient populations and definition of low-level viremia were divergent from our studies. Other studies have demonstrated that HIV-RNA plasma concentrations decrease faster in the first weeks after initiating treatment Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 with INSTI-containing ART compared to PI class antiretrovirals [6,18–20] or EFV [21]. As the vast majority of patients were receiving RAL, further investigation on other INSTI agents, such as DTG, are needed. In contrast to several previous studies [13,14,22,23], no effect of NNRTI on residual viremia emerged in our study. This discrepancy could be explained by differences in NNRTI agents, while patients in our cohort were more commonly treated with rilpivirine (n=216) and efavirenz (n=188) and less so with nevirapine (n=4). Based on ultralow virological suppression, our findings tend to support recommendations of current treatment guidelines to start combined ART with an INSTI-based regimen, independent of CD4+ count and HIV-RNA VL [3,4]. Since persistent residual viremia appears to impact immune activation, inflammation and microbial translocation [24,25], VLs at ultralow levels could bear some clinical importance. The ongoing presence of virus, despite low viral concentrations, could prevent some systemic inflammatory markers from returning to normal levels; however, the association between residual inflammation and residual HIV-1 replication is probably bi-directional and complex [26]. Indeed, as shown in several studies among patients undergoing treatment intensification [27–31] or effective ART for seven years [27], residual viremia under ART has been associated with low-grade immune activation. Another study has shown that immune activation still persists despite UL-VL not detected after 10 years of treatment [28], but was assessed with only one cross-sectional measure of UL-VL. Several studies have shown that restoration of the CD4/CD8 ratio is associated with faster virological control after ART initiation (VL <50 copies/mL) [29,30]. At the same time, CD4 decline appears to be accelerated in studies investigating immunologic progression among elite HIV controllers without ART [29–31]. In our study, we observed that patients with more extensive viral suppression below ultrasensitive detection levels during ART, defined as ≥50% of follow-up with Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 UL-VL not detected, have faster increases of CD4 cell count and CD4/CD8 ratio. Interestingly, CD4/CD8 ratio is inversely correlated to levels of inflammation and immune activation in patients with virological suppression [32]. Whether the immunological effects linked to increased UL-VL suppression also corresponds to improvement in inflammation remains to be fully elucidated. The relationship between HIV-RNA replication and VR has been examined in several studies using assays with higher detection thresholds, but this association with respect to residual viremia is unclear. Past research has shown that patients with slower time to achieving <50 copies/mL are more likely to continue treatment with residual viremia [13] and, when <50 copies/mL occurs after 6 months from initiating ART, have an almost 2-fold risk of subsequent VR [6]. Reports with clearer evidence state that the inability to achieve UL-VL during first-line therapy could lead to a higher risk of VR in certain settings [33,34], while others fail to observe similar results [35]. In our study, we observed that patients without VR were able to suppress UL-VL not detected at a significantly faster rate, similar to patients with “high” suppression of UL HIV-RNA during the course of treatment. Furthermore, we demonstrated that the longer UL-VL not detected occurred during treatment duration, the lower the risk of VR. These results suggest the importance of extensive long-term suppression in order to avoid subsequent VR. Our study has several noteworthy advantages. The large number of patients with many repeated samples, including HIV-RNA VL measures, is one of the most extensive to date. These data allow more flexibility in analytical options, hence why we decided to examine not only proportion of viral suppression during follow-up, as have others [13], but more appropriately, the risk factors associated with the rate of ultralow viral suppression across all visits over time. This is an important distinction as proportion of viral suppression can be biased by length of follow-up and most studies have evaluated this statistic at a single time-point (i.e. last follow-up visit). Furthermore, we did not Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 focus only on first-line ART regimens or time until first VR, but rather the overall therapeutic strategies adapted throughout follow-up (by including time-varying treatment switches in the models) and the multiple rounds of VR (via conditional risk-set proportional hazards models). Including this information provides a more accurate representation of the dynamic processes occurring during routine follow-up. Despite these advantages, our study has some limitations. First, this study lacks information on adherence, pharmacological data and ART-associated adverse events, which could play a role in UL- VL detection [36,37]. Second, we did not analyze data on genotypic resistance at the start of treatment or at the time of rebound. Third, HIV-RNA VLs were measured using a commercially- available assay as part of clinical follow up. Single Copy Assays are more adapted to measuring ultrasensitive detection; nevertheless, this time- and cost-prohibitive assay requires large amounts of blood to be collected and cannot be readily used in routine practice [34]. The major limitation of routine PCR is that when HIV RNA VLs are extremely low, within-assay variation is large and could have an impact on reproducibility when evaluating detection or within-patient variation [38]. It should be mentioned, however, that other studies investigating residual viremia have also used real- time PCR assays with fairly reproducible conclusions [10,13,17,33]. Finally, although estimates were adjusted for the number of tests per year, we cannot completely exclude missed virological rebounds. Finally, PI-containing regimens were more often given to individuals with high HIV-RNA VL and low CD4+ T-cell count at ART-initiation and this bias could have influenced our results. Although we did adjust for these factors in multivariable analysis, residual confounding cannot be fully excluded. In conclusion, notwithstanding the non-randomized allocation of treatment combinations, we showed that diverse ART regimens have different effects on residual viremia after achieving <50 Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 HIV-RNA copies/mL. In particular, higher rates of virological suppression with UL-VL not detected are more common in individuals undergoing INSTI-based ART regimens. Moreover, given the association between residual viremia and immune activation, INSTI class antiretrovirals, a preferred third agent for first-line ART according to current recommendations, could also lead to improvement in immunological parameters and possibly reduced inflammation. Further evidence would be needed, however, on other INSTI class agents to confirm this hypothesis. FUNDING This work was supported by the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales. TRANSPARENCY DECLARATIONS S.L has received travel grants from Gilead Sciences and ViiV Healthcare. JLM has received travel grants from Abbvie Labs, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare and has received honoraria for invited talks from Gilead Sciences, Merck Laboratories MSD and ViiV Healthcare. PMG has received grant support from BMS and Janssen Pharmaceuticals, personal fees as member of symposia faculty from BMS, Abbvie Labs, and Mylan and honoraria for participation on international boards from Gilead Sciences and ViiV Healthcare. CK has received grant support from Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. A. G. M. has received honoraria for advisories or invited talks or conferences and research grants from Abbvie Labs, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. LMJ have received honoraria from BMS, Mylan, Gilead Sciences, Merck Laboratories MSD, Janssen Pharmaceuticals and ViiV Healthcare. The other authors have none to declare. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 REFERENCES 1. Palmer S, Maldarelli F, Wiegand A, et al. 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Characterizing Patients with Very- Low-Level HIV Viremia: A Community-Based Study. J Int Assoc Provid AIDS Care 2017; 16:261–266. 36. Leierer G, Grabmeier-Pfistershammer K, Steuer A, et al. Factors Associated with Low-Level Viraemia and Virological Failure: Results from the Austrian HIV Cohort Study. PloS One 2015; 10:e0142923. 37. Pasternak AO, de Bruin M, Jurriaans S, et al. Modest nonadherence to antiretroviral therapy promotes residual HIV-1 replication in the absence of virological rebound in plasma. J Infect Dis 2012; 206:1443– 38. Ruelle J, Debaisieux L, Vancutsem E, et al. HIV-1 low-level viraemia assessed with 3 commercial real- time PCR assays show high variability. BMC Infect Dis 2012; 12:100. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 TABLES Table 1. Description of the study population at antiretroviral therapy initiation Initial ART backbone NRTI+NNRTI NRTI+PI NRTI+INSTI N=717 (n=211) (n=419) (n=87) p Male/Female (% males) 527/190 (73.5) 171/40 (81.0) 293/126 (69.9) 63/24 (72.4) 0.01 Age* [N=716] 39 (32-46) 39 (32-47) 38 (32-45) 42 (34-51) 0.009 Hospital 0.005 La Pitié-Salpétrière 519 (72.4) 160 (75.8) 286 (68.3) 73 (83.9) Saint-Antoine 198 (27.6) 51 (24.2) 133 (31.7) 14 (16.1) Time since first positive HIV serology*, years 0.2 (0.1-2.4) 0.5 (0.1-2.5) 0.2 (0.1-2.3) 0.1 (0.1-2.9) 0.2 AIDS-defining illness 103 (14.4) 16 (7.8) 75 (17.9) 12 (13.8) 0.002 CD4+ T-cell count*, /mm [N=666] 306 (178-441) 366 (268-467) 275 (137-408) 322 (210-408) <0.001 Nadir CD4+ T-cell count*, mm 275 (157-374) 328 (242-410) 238 (110-349) 278 (168-360) <0.001 CD8+ T-cell count*, /mm [N=643] 780 (538-1141) 822 (586-1241) 764 (497-1115) 780 (564-1167) 0.10 CD4:CD8 ratio* [N=643] 0.35 (0.21-0.57) 0.41 (0.24-0.63) 0.31 (0.17-0.51) 0.35 (0.21-0.57) <0.001 HIV-RNA viral load*, log copies/mL 4.84 (4.36-5.24) 4.68 (4.17-5.08) 4.90 (4.41-5.33) 4.84 (4.49-5.29) <0.001 HIV-RNA viral load >10 copies/mL 292 (40.7) 66 (31.3) 187 (44.6) 39 (44.8) 0.004 HIV subtype [N=105] 0.7 B 43 (41.0) 12 (48.0) 25 (37.3) 6 (46.2) CRF02_AG 34 (32.4) 6 (24.0) 25 (37.3) 3 (23.1) Other 28 (26.7) 7 (28.0) 17 (25.4) 4 (30.8) Number of antiretroviral agents* 3 (3-3) 3 (3-3) 3 (3-3) 3 (3-3) 0.6 Positive HCV RNA [N=291] 18 (6.2) 4 (5.5) 7 (3.9) 7 (18.9) 0.002 HCV RNA viral load, log IU/mL* 6.20 (5.70-6.55) 6.20 (5.52-6.56) 6.55 (5.88-6.71) 6.15 (5.63-6.39) 0.5 Positive HBsAg serology [N=705] 23 (3.3) 5 (2.4) 18 (4.3) 0 (0) 0.09 Switching ART regiment during follow-up 425 (59.3) 92 (43.6) 284 (67.8) 49 (56.3) <0.001 Number of switches during follow-up* 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 0.8 All statistics are n (%), except for continuous variables (*) where medians (IQR) are reported. † ‡ Only among patients with positive HCV RNA viral loads. Only among patients switching ART regiments at least once. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Table 2. Determinants of UL-VL not detected during antiretroviral therapy Univariable Multivariable* IRR (95%CI) p IRR (95%CI) p Age (per 10 years) [N=716] 0.88 (0.83-0.92) <0.001 0.89 (0.85-0.94) <0.001 Gender Male 1.00 1.00 Female 1.13 (1.00-1.28) 0.06 1.14 (1.01-1.29) 0.04 Baseline HIV-RNA <10 copies/mL 1.00 1.00 ≥10 copies/mL 0.67 (0.60-0.75) <0.001 0.72 (0.64-0.80) <0.001 Baseline CD4+ T-cell count [N=665] >500 cells/mm 1.00 1.00 350-500 cells/mm 0.91 (0.77-1.08) 0.3 0.90 (0.76-1.06) 0.2 <350 cells/mm 0.63 (0.54-0.74) <0.001 0.66 (0.57-0.77) <0.001 Baseline CD4+ T-cell nadir ≥250 cells/mm 1.00 <250 cells/ mm 0.68 (0.61-0.76) <0.001 Time-varying CD4:CD8 ratio [N=681] ≥0.8 1.00 <0.8 0.89 (0.81-0.99) 0.03 Anchor drug of ART during follow-up NNRTI 0.99 (0.91-1.07) 0.8 0.99 (0.91-1.07) 0.8 PI 0.98 (0.91-1.07) 0.7 0.96 (0.89-1.05) 0.4 Integrase inhibitor 1.13 (1.01-1.25) 0.03 1.15 (1.04-1.28) 0.009 Other combination 0.91 (0.79-1.06) 0.2 0.91 (0.78-1.06) 0.2 Concomitant treatment switch No 1.00 Yes 0.97 (0.86-1.09) 0.6 Analysis includes 717 HIV-infected patients initiating antiretroviral therapy (ART) and accounts for subsequent lines of ART after switching regimens. Incident rate ratios (IRR) and 95% confidence intervals (CIs) calculated from a random-effect Poisson regression accounting for patient variability at baseline. *In multivariable analysis, baseline nadir CD4+ was closely linked to baseline CD4+ T-cell count and hence excluded. Time-varying CD4:CD8 ratio (preferred over CD4:CD8 ratio at baseline) was excluded because its associated p-value was below the pre-specified threshold (p=0.402). A total of 52 patients had missing data on baseline CD4 T-cell count (n=51) or age (n=1) and were not included the multivariable model. Individual ARV classes are compared to all classes combined as a reference group. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Table 3. Determinants of virological rebound after antiretroviral therapy initiation Multivariable** Multivariable*** Univariable* Model 1 Model 2 HR (95%CI) p HR (95%CI) p HR (95%CI) p 0.95 (0.80- Age (per 10 years) [N=716] 1.14) 0.6 Gender Male 1.00 0.72 (0.46- Female 1.11) 0.14 Baseline HIV-RNA <10 copies/mL 1.00 1.00 1.00 1.43 (0.99- 1.45 (0.96- 1.18 (0.78- ≥10 copies/mL 2.09) 0.06 2.19) 0.08 1.79) 0.4 Cumulative duration with UL-VL 0.41 (0.29- 0.45 (0.30- not detected 0.58) <0.001 0.67) <0.001 Baseline CD4+ T-cell count [N=666] >500 cells/mm 1.00 1.51 (0.83- 350-500 cells/mm 2.74) 0.18 1.25 (0.71- <350 cells/mm 2.19) 0.4 Baseline CD4+ T-cell nadir ≥250 cells/mm 1.00 0.86 (0.57- <250 cells/mm 1.30) 0.5 Time-varying CD4:CD8 ratio [N=681] ≥0.8 1.00 1.00 1.00 2.04 (1.32- 1.95 (1.17- 1.87 (1.15- <0.8 3.18) 0.001 3.24) 0.01 3.05) 0.01 Anchor drug of ART during follow- up 0.60 (0.43- 0.76 (0.50- NNRTI 0.84) 0.003 1.15) 0.2 - 1.20 (0.88- 1.00 (0.69- PI 1.64) 0.2 1.43) 0.9 - 0.82 (0.48- 1.23 (0.64- Integrase inhibitor 1.40) 0.5 2.33) 0.5 - 1.68 (1.11- 1.08 (0.66- Other combination 2.56) 0.02 1.75) 0.8 - Concomitant treatment switch No 1.00 1.48 (0.94- Yes 2.32) 0.09 Number of HIV-RNA tests per year ≤3 0.33 (0.18- <0.001 0.29 (0.14- 0.002 0.24 (0.12- <0.001 Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 0.62) 0.63) 0.49) 0.78 (0.39- 0.83 (0.36- 0.66 (0.31- 3-4 1.58) 0.5 1.93) 0.7 1.40) 0.3 1.25 (0.63- 1.29 (0.61- 0.99 (0.50- 4-6 2.50) 0.5 2.71) 0.5 1.95) 0.9 >6 1.00 1.00 1.00 Analysis includes 717 HIV-infected patients initiating antiretroviral therapy (ART) and accounts for subsequent lines of ART after switching regimens. Hazard ratios (HR) and 95% confidence intervals (CIs) were estimated from a conditional risk-set, Cox proportional hazards model (using time from the previous event). *Adjusted for number of HIV-RNA tests per year to account for individuals with more frequent testing (hence, higher possibility of detecting VR). **In the multivariable model, treatment switch was excluded from the model as its associated p-value was below the pre-specified threshold (p=0.157). A total of 36 patients had missing data on CD4:CD8 ratio and were not included in the multivariable model. ***Replacing cumulative duration with UL-VL not detected as a time-varying variable with ART regimen in Model 1 (given the collinearity between these two variables). Individual ARV classes are compared to all classes combined as a reference group. Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 FIGURE LEGENDS Figure 1. Time to reach UL-VL not detected and changes in immunological parameters after initiating antiretroviral therapy according to residual viremia and virological rebound Cumulative proportion of patients reaching an undetectable UL-VL with detection threshold at ultrasensitive levels is depicted in (A), while adjusted for baseline HIV-RNA viral load. Locally- weighted scatterplot smoothing lines are constructed for CD4+ T-cell count (B) and CD4+:CD8+ ratio (C) during follow-up. All figures are stratified on patients with low versus high HIV-RNA suppression (defined as <50% versus ≥50%, respectively, of follow-up spent with an UL-VL not detected). Accepted Manuscript Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofz177/5435897 by Ed 'DeepDyve' Gillespie user on 16 April 2019 Figure 1. 1.00 0.80 p<0.001 0.60 0.40 0.20 L ow US suppr High US suppr 0.00 0 1 2 3 4 5 6 T ime (years ) p=0.03 L ow US s uppr High US s uppr 0 1 2 3 4 T ime (years ) 1.2 1.0 0.8 p=0.001 0.6 0.4 0.2 L ow US suppr High US suppr 0.0 0 1 2 3 4 T ime (years ) Accepted Manuscript C umulative probability C D4+ cell count (/mm3) C D4+:C D8+ ratio

Journal

Open Forum Infectious DiseasesOxford University Press

Published: May 1, 2019

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