The CD4:CD8 ratio is associated with markers of age-associated disease in virally suppressed HIV-infected patients with immunological recovery

Objectives Inversion of the CD4:CD8 ratio (< 1) has been identified as a hallmark of inmmunosenescence and an independent predictor of mortality in the general population. We aimed to assess the association between the CD4:CD8 ratio and markers of age-associated disease in treated HIV-infected patients with good immunovirological response. Methods A cross-sectional analysis was conducted in 132 HIV-infected adults on antiretroviral therapy (ART), with plasma HIV RNA < 50 HIV-1 RNA copies/mL for at least 1 year, CD4 count > 350 cells/μL and age < 65 years. We analysed the associations between the CD4:CD8 ratio and subclinical atherosclerosis [assessed using carotid intima-media thickness (IMT)], arterial stiffness [assessed using the augmentation index (AIx)], the estimated glomerular filtration rate (eGFR), muscle wasting and sarcopenia [assessed using appendicular lean mass/height 2 (ALM) measured by dual-energy X-ray absorptiometry (DEXA)]. Results CD4: CD8 ratio inversion was associated with higher IMT, lower eGFR and lower ALM (all values P < 0.05), but not with AIx. In multivariate analyses adjusted for age, sex, hypertriglyceridaemia, tobacco use and cumulative ART exposure, inversion of the CD4:CD8 ratio was independently associated with higher IMT [odds ratio (OR) 2.9; 95% confidence interval (CI) 1.2-7.1], arterial stiffness (OR 4.8; 95% CI 1.0-23.5) and lower eGFR (OR 5.2; 95% CI 1.0-64.4), but not sarcopenia (OR 0.7; 95% CI 0.2-2.7). These associations persisted when models were applied to subjects with nadir CD4 counts > 200 cells/μL and those with CD4 counts > 500 cells/μL. Conclusions The CD4:CD8 ratio in treated HIV-infected subjects with good immunovirological response is independently associated with markers of age-associated disease. Hence, it might be a clinically useful predictor of non-AIDS-defining conditions. Keywords: carotid intima-media thickness, CD4:CD8 ratio, HIV, premature aging, sarcopenia, vascular stiffness Accepted 24 July 2013 Introduction Antiretroviral therapy (ART) is among the greatest successes of modern medicine, having rapidly changed the prognosis of HIV-infected individuals from years to decades of ORIGINAL RESEARCH 1 survival. However, ART has failed in its attempt to completely restore normal health to HIV-infected subjects. Although the reasons remain poorly understood, subjects on successful ART still present increased morbidity and mortality relative to uninfected individuals Methods Study design, participants, setting and eligibility We conducted an observational, cross-sectional study of 132 consecutive HIV-infected patients who attended a university-based HIV clinic in Madrid between February and October 2011. Subjects were recruited if they were HIV infected and on stable triple ART, defined as continuous treatment with three antiretroviral drugs including either a nonnucleoside reverse transcriptase inhibitor or a protease inhibitor, had had undetectable plasma HIV RNA levels for at least 1 year, and showed CD4 counts ≥ 350 cells/uL at inclusion in the study. As we aimed to analyse variables associated with subclinical aging, exclusion criteria included age > 65 years and the presence of cardiovascular disease (previous stroke, myocardial infarction or intermittent claudication) or chronic kidney disease. The study conformed to the principles of the Declaration of Helsinki and the Good Clinical Practice Guidelines and was approved by the local Ethics Committee. All patients gave their written informed consent to participate in the study. Clinical and laboratory measurements Medical records were carefully reviewed and all subjects underwent a physical examination. Information on gender, age, body mass index, smoking status, family history of cardiovascular disease and treatment with antiretroviral drugs was recorded. The presence of arterial hypertension, hypercholesterolaemia and hypertriglyceridaemia was defined according to the Adult Treatment Panel III criteria © 2013 British HIV Association HIV Medicine Renal function assessment We used the recently developed Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula to calculate the estimated glomerular filtration rate (eGFR), which has proved to be more accurate than the routinely used Modification of Diet in Renal Disease (MDRD) formula, especially in patients with normal kidney function Measurement of subclinical atherosclerosis The degree of subclinical atherosclerosis was evaluated by means of the carotid intima-media thickness (IMT), which has been demonstrated to be an important predictor of cardiovascular events in both the general population Measurement of arterial stiffness The augmentation index (AIx) was used as a surrogate marker of arterial stiffness, which correlates with cardiovascular risk Statistical analysis Qualitative variables were summarized as a frequency distribution and normally distributed quantitative variables as mean ± standard deviation. Continuous nonnormally distributed variables were summarized as median and interquartile range (IQR). Means for variables with a normal distribution were compared using the t-test. Nonparametric variables were assessed using the Mann−Whitney test. Given the nonnormal distribution of some of the variables, the Spearman correlation coefficient was used to analyse the correlation between continuous variables. Independent associations between the CD4:CD8 ratio [as a binary variable: an inverted CD4:CD8 ratio (< 1) vs. a normal CD4:CD8 ratio (≥ 1)] and binary outcomes of subclinical aging (IMT > or ≤ p50, presence or absence of arterial stiffness as described above, eGFR > or ≤ p50, and presence or absence of sarcopenia as described above) were evaluated in a multivariate analysis. The modelling strategy involved explanatory logistic regression analysis. Variables with an imbalance between the study groups were introduced into the model, as well as those variables that could exert confounding on the association between the CD4:CD8 ratio and the dependent variables. The variables nadir CD4 and CD8 T-cell counts were excluded from the models because of collinearity. Hence, the maximum model was adjusted by age, sex, hypertriglyceridaemia, tobacco use and cumulative ART exposure. Cumulative tenofovir exposure was also included in the model for renal impairment. As arterial stiffness was defined according to sex and age reference values and sarcopenia according to sex, these demographic variables were not included in the multivariate model for each of these two dependent variables. A backward strategy was used, considering a significance HIV, CD4:CD8 ratio and subclinical aging 3 © 2013 British HIV Association HIV Medicine (2013) level of 0.05 to eliminate variables. The overall model was reapplied including only the confounding factors that, after their elimination from the model, produced a ≤ 10% change in the estimate of the coefficient of the variable of principal exposure (inverted CD4:CD8 ratio). The magnitude of association was evaluated using the odds ratio (OR) and 95% confidence interval (CI). In order to assess whether the CD4:CD8 ratio may provide additional information to CD4 nadir and CD4 counts, we applied the multivariate models to the subgroups of patients with low nadir CD4 counts (< 200 cells/μL) and high CD4 counts (> 500 cells/ μL). The null hypothesis was rejected with a type I error < 0.05 (α < 0.05). Statistical analyses were performed using STATA version 12.0 (StataCorp LP, College Station, TX), and figures were generated using GRAPHPAD PRISM 5.00 (GraphPad Software, San Diego, CA). Results All variables assessing subclinical aging -IMT and arterial stiffness for cardiovascular aging, eGFR for renal impairment and ALM as a marker of muscle wasting and sarcopenia -are summarized in Differences in demographic and immunovirological variables, cardiovascular risk factors and inflammation biomarkers according to the CD4:CD8 ratio Associations between subclinical aging and the CD4:CD8 ratio Next, we explored the linear and categorical associations between the CD4:CD8 ratio and variables associated with subclinical aging, reflecting carotid atherosclerosis (IMT), arterial stiffness (AIx), renal impairment (eGFR) and muscle wasting or sarcopenia (ALM). First, we analysed the linear correlations between the CD4:CD8 ratio and IMT, eGFR and ALM Multivariate analysis A series of logistic regression models were explored to analyse the association of the CD4:CD8 ratio with variables reflecting subclinical aging (IMT, arterial stiffness, eGFR and sarcopenia), which were used as dependent variables in consecutive multivariate models, adjusting by age, sex, hypertriglyceridaemia, tobacco use and cumulative ART exposure (and specifically tenofovir exposure in the model for renal impairment). In the multivariate analyses, no associations with sarcopenia were observed, but subjects with inversion of the CD4:CD8 ratio showed a significant threefold increased risk of higher IMT and a significant fivefold increased risk of arterial stiffness and lower eGFR Discussion In this study in virally suppressed, HIV-infected subjects, we explored the associations of the CD4:CD8 ratio with markers of vascular disease, kidney disease and muscle wasting. We found that the CD4:CD8 ratio was negatively correlated with IMT, and positively correlated with eGFR (both values, P < 0.05). In addition, subjects with inversion of the CD4:CD8 ratio more frequently showed higher IMT, lower eGFR and lower muscle mass (all values, P < 0.05). No significant associations were detected with arterial stiffness. In multivariate analyses, inversion of the CD4:CD8 ratio was independently associated with higher IMT, arterial stiffness and lower eGFR. Importantly, these associations remained statistically significant when the sample was censored by low nadir CD4 count (< 200 cells/μL) and low CD4 T-cell count (< 500 cells/μL), suggesting that the CD4:CD8 ratio provides additional information to that provided by nadir CD4 and CD4 T-cell counts as surrogate markers of age-associated disease 6 S Serrano-Villar et al