Antiretroviral therapy (ART) prevents AIDS-related morbidity and mortality1–3 and reduces HIV transmission.4 In addition, ART reduces systemic inflammation,5–7 immune activation,8 and coagulopathy9 by achieving and sustaining viral suppression but only partially to levels observed in HIV-negative individuals.6,10,11 This chronic residual inflammation and coagulopathy have been linked to the development of non-AIDS complications, including cardiovascular disease, cancer, and death.12–19 Although multiple interventions aimed at improving residual inflammation have been evaluated (eg, ART intensification, anti-inflammatories, treating coinfections),20–25 most have shown modest or no beneficial effect. Thus, effective strategies to reduce residual inflammation in treated HIV infection are needed.26
Sustained ART adherence is required to achieve durable virological suppression, yet the relationship between adherence and viral suppression is complex and dynamic.27–30 Perfect (ie, 100%) adherence is not required to achieve or sustain viral suppression, and viral suppression is not necessarily a perfect surrogate for complete adherence (ie, ART can be interrupted for short periods without the development of viremia using conventional assays).31–35 However, the consequences of suboptimal adherence, beyond suppression, are unknown. Recently, low self-reported adherence was associated with higher levels of residual inflammation and immune activation in chronically suppressed men living with HIV.36 This association has not been replicated nor has been evaluated in women, treatment-naive individuals, or in cohorts using objective measures of ART adherence. To address this gap, we aimed to determine whether ART adherence, measured by electronic monitoring, is associated with biomarkers of systemic inflammation and coagulopathy among treatment-naive individuals living with HIV who initiate nonnucleoside reverse transcriptase inhibitors (NNRTI) and thymidine analog–based ART.
We evaluated treatment-naive adults living with HIV who initiated first-line ART between 2005 and 2010 and were enrolled in the Uganda AIDS Rural Treatment Outcomes cohort (UARTO, NCT01596322) at a regional referral hospital in Mbarara, Uganda.37–39 In UARTO, participants were followed every 3–4 months; blood was collected for plasma and cell isolation, including CD4+ T-cell count and HIV viral load (VL; Amplicor HIV Monitor 1.5 test; Roche, Branchburg, NJ), at baseline and subsequent visits. For this analysis, we evaluated participants who (1) had available biomarker levels at baseline and after 6 (±1) months of ART; (2) had HIV VL <400 copies per milliliter at the 6-month visit, and; (3) had available ART adherence data for at least 3 months in the 6-month period.
ART adherence (across the 6-month study period) was measured using the medication event monitoring system (MEMS) electronic pill bottle (Aardex Group, Sion, Switzerland), which recorded the date and time for each bottle opening. Average ART adherence was calculated based on the number of observed cap openings divided by the number of prescribed doses/day in the 6-month period (capped at 100%).
Biomarkers of Inflammation, Coagulopathy, and CD8+ T-Cell Activation
Plasma was centrifuged and stored at −80°C until analysis. Most (95%) samples were stored in acid citrate dextrose, whereas the remaining in EDTA; to account for this difference, an adjustment factor of 1.276 was used for biomarkers tested from acid citrate dextrose tubes.39 D-dimer (Diagnostico Stago, Parsippany, NJ), interleukin 6 (IL-6; Human IL-6 Ultra-Sensitive Kit; Meso Scale Diagnostics, Rockville, MD), soluble (s)CD14 (sCD14; R&D Systems, Minneapolis, MN), sCD163 (sCD163; Trillium Diagnostics, Bangor, ME), and the kynurenine/tryptophan ratio were measured in thawed plasma samples, as previously reported.38,39 The percentage of human leukocyte antigen-D–related (HLA-DR)+/CD38+ CD8+ T cells was measured in fresh whole-blood specimens processed on the day of collection, as previously described.37
Demographic and baseline cohort characteristics were summarized. Biomarker concentrations were log transformed, except the proportion of HLA-DR+/CD38+/CD8+ T cells, which was analyzed as an absolute value. The crude change in biomarkers and CD8+ T-cell activation between baseline and the 6-month visit was analyzed using paired t tests. Average ART adherence was considered to be continuous. We used scatter plots to graphically evaluate the relationship between average ART adherence and the outcomes of interest at the 6-month visit, assessing for a linear relationship between the explanatory and outcome variables. We then fit linear regression models to estimate the change in biomarkers and CD8+ T-cell activation after 6-month on ART with changes in ART adherence, adjusting for baseline biomarker values. We initially evaluated a model where average ART adherence was the primary predictor of interest. We then used a model that adjusted for potential confounders, including age, gender, CD4+ T-cell count, baseline HIV VL, depression (using the Hopkins symptoms checklist score40,41), and heavy alcohol use (using the Alcohol Use Disorders Identification Test-C questionnaire42). Variables excluded from the unadjusted model because of missing data and/or collection later during the study included smoking status, illicit drug use, food security, body mass index, and anemia. Because biomarkers of inflammation and CD8+ T-cell activation were analyzed as related and complementary outcomes, we did not correct for multiple comparisons.43 We also performed sensitivity analyses, limiting our sample to participants with an HIV VL of <40 copies per milliliter at the 6-month visit (as the HIV VL assay evolved throughout the course of the study) and removing high-influence observations with DFBETA values outside the range of ±2/sqrt (n) or a Cook D value of >1.0. Statistical analyses were performed using SAS 9.4 (SAS Institute, Inc, Cary, NC). A P value of <0.05 was considered to be statistically significant.
Study procedures were reviewed and approved by the Institutional Review Boards of the Mbarara University of Science and Technology and Partners Healthcare/Massachusetts General Hospital and the Ugandan National Council of Science and Technology. All participants provided written informed consent.
Of a total of 546 participants enrolled in the UARTO cohort during the study period, 282 met our evaluation criteria for at least 1 outcome and were included in the unadjusted analysis. Reasons for exclusion were as follows: baseline visit only (24%), no 6-month visit within study window (8%), <3 month MEMS data (8%), no ART initiation within study window (8%), and no HIV VL or HIV VL of >400 copies per milliliter at 6-month visit (4%). The median age was 35 years [interquartile range (IQR) 30–40 years], and 196 (70%) of the participants were women. Heavy alcohol use and depression were reported at least once in 39 (15%) and 92 (34%) of participants, respectively. Most ART regimens were NNRTI based [244 (89%) nevirapine and 21 (7%) efavirenz], with a nucleoside reverse transcriptase inhibitor backbone consisting of zidovudine/lamivudine (68%), stavudine/lamivudine (28%), or other (4%). Median CD4+ T-cell count was 134 cells per cubic millimeter (IQR, 80–198 cells/mm3), and 54% of participants had a baseline HIV VL of >100,000 copies per milliliter. Baseline characteristics of virologically suppressed participants who were included in the adjusted analysis vs. those excluded because of missing data are presented in Table 1.
ART Adherence and Biomarkers of Inflammation and Coagulopathy
The median ART adherence was 93% (IQR, 84%–98%); 13 (5%) participants had a mean 6-month adherence of 100%, 194 (68%) had an adherence of 85%–100%, and 75 (27%) had an adherence of <85%. We observed a significant decrease in all biomarkers between the pre-ART and 6-month visits (P < 0.0001 for all biomarkers, except sCD14 P = 0.003). In the analyses adjusted for baseline values of biomarkers only, we identified a statistically significant inverse linear relationship between average ART adherence and biomarkers of inflammation and coagulopathy (Supplemental Digital Content Table 1, http://links.lww.com/QAI/B109, Fig. 1). After additionally adjusting for age, gender, a positive depression screen, heavy alcohol use, and baseline CD4+ T-cell count, and HIV VL (Table 2), these relationships remained statistically significant for IL-6 (15% decrease; P < 0.0001), D-dimer (10% decrease; P = 0.017), and sCD14 (3% decrease; P = 0.028) in participants who achieved virologic suppression to <400 copies per milliliter at the 6-month visit. In the adjusted model for participants who achieved virologic suppression to <40 copies per milliliter, IL-6 (11% decrease; P = 0.040) and sCD163 (7% decrease; P = 0.009) remained statistically significant, despite a smaller sample size (Table 2). These findings were similar (and the slopes in all biomarkers remained negative) when removing highly influential observations (Supplemental Digital Content Table 2, http://links.lww.com/QAI/B109). A sensitivity analysis adjusting for the most prevalent ART regimens (limited to participants on 3 TC/AZT/NVP or 3 TC/d4T/NVP, which corresponded to 88% of the ART regimens in this cohort) did not show any additional effect on biomarker levels (data not shown).
We demonstrated an inverse relationship between ART adherence, measured using electronic monitoring, and plasma concentrations of biomarkers of inflammation and coagulopathy in treatment-naive Ugandan adults who achieved virologic suppression after 6 months of ART. These relationships remained significant for IL-6, D-dimer, and sCD14 after adjusting for several covariates, including pre-ART CD4+ T-cell count and VL, with decreases of 3%–15% in each biomarker for each 10% increase in average adherence. Interestingly, our observations were the strongest for IL-6, which has also been found to be the strongest predictor of adverse outcomes in comparison with other markers16 and could have been influenced by our overall low CD4 T cell at baseline, as has been previously proposed.44 To our knowledge, this study is the first to demonstrate a relationship between electronically monitored adherence using MEMS, inflammation, and coagulopathy among virologically suppressed patients. Collectively, these findings suggest that variations in ART adherence could have biological consequences that extend beyond achieving and sustaining virologic suppression.
Our findings are consistent with previous observations, where <100% ART adherence (measured subjectively through self-report) was associated with higher levels of inflammatory biomarkers in virologically suppressed men on ART in the United States.36 Among the possible explanations for our findings is that suboptimal ART adherence could lead to low-level viral replication below the limit of detection of most clinically available assays,45–47 which may result in spurts of inflammation and immune activation.48,49 Likewise, incomplete ART adherence could also be associated with intermittent episodes of viremia that are not captured between visits. Further research to evaluate these and other possible mechanisms is needed.
The findings in this study could have clinical implications that deserve further evaluation. Given the relationship between low ART adherence and higher levels of IL-6, which has been associated with higher morbidity and mortality in HIV,12,15,16 suboptimal ART adherence could conceivably also be associated with worse clinical outcomes that extend beyond those prevented by sustained virologic control, although this relationship remains unknown. It is also unclear whether strategies to improve ART adherence can reduce chronic residual inflammation and its downstream consequences in treated HIV infection. Interestingly, in a recent clinical trial of ART intensification among individuals maintaining plasma HIV RNA levels of <40 copies per milliliter, a significant reduction of at least 1 biomarker of immune activation (activated CD4+ T cells) was observed in the placebo arm.23 This was coupled with an early reduction in low-level viremia using a highly sensitive single-copy assay,23 which was also reported in the placebo arm of a second intensification study.24 Although the mechanisms behind these findings in the participants randomized to placebo remain unclear, it is plausible that they could have been mediated, at least partially, by an improvement in ART adherence after enrollment in a clinical trial (ie, Hawthorne effect). Although any single intervention is unlikely to completely reverse residual inflammation and its clinical consequences, ART adherence could play a significant synergistic role to achieve this goal. Future studies evaluating the impact of adherence optimization beyond virologic suppression are necessary to corroborate these hypotheses.
Among the strengths of our study are the inclusion of a diverse population with a large proportion of women in resource-limited settings, the use of an objective adherence measure that is more informative than self-report,50 and the inclusion of multiple biomarkers of systemic inflammation, innate and acquired immune activation, and coagulopathy. The main limitations include the use of a relatively high VL cutoff (<400 copies/mL), the evaluation of primarily older NNRTI-based ART regimens, and the potential influence of unmeasured confounders (ie, smoking and diet). Further studies to determine if these findings persist with even lower VL thresholds and during long-term virologic control are needed.
In addition, these findings should be replicated in the era of modern ART, to determine if the relationships between adherence and inflammation in suppressed patients are generalizable to those taking more forgiving antiretrovirals, such as the integrase strand transfer inhibitors.
In summary, we demonstrated that lower ART adherence is associated with higher inflammation and coagulopathy in treatment-naive Ugandans who achieved virologic suppression after 6 months of therapy. These findings confirm previous observations and suggest that optimal adherence may be required to maximize the biological benefit or ART.
The authors thank the UARTO participants and staff who made this study possible.
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