Prevalence of Silent Atherosclerosis and Other Comorbidities in an Outpatient Cohort of Adults Living with HIV: Associations with HIV Parameters and Biomarkers

Abstract

People living with HIV (PLWH) are at risk of noninfectious comorbidities. It is important to individualize those at higher risk. In a single-center cohort of PLWH, we performed a cross-sectional analysis of comorbidities, diagnosed according to standard procedures. The primary endpoint was the prevalence of subclinical carotid/coronary atherosclerosis. Secondary endpoints were its association with selected inflammatory/immune activation biomarkers and with other comorbidities. Associations were examined by using Chi-square or Fisher's exact test for categorical variables and Student or Wilcoxon tests for quantitative variables, and a stepwise multivariate logistical model was performed for further exploration. Among 790 participants [median age: 49.8 years (interquartile range, IQR: 44.5–55.6), 77.1% males, median CD4: 536/mm³ (IQR: 390–754), 83.6% with undetectable viral load], asymptomatic atherosclerosis was found in 26% and was associated in multivariate analysis with older age, longer known duration of infection, higher sCD14, and lower adiponectin levels. Hypertension was found in 33.5% of participants, diabetes in 19.4%, renal impairment in 14.6%, elevated low-density lipoprotein-cholesterol in 13.3%, elevated triglyceride/high-density lipoprotein (HDL)-cholesterol ratio in 6.6%, and osteoporosis in 7.9%. The presence of two or more comorbidities was found in 42.1% of participants and was associated in multivariate analysis with older age and longer exposure to antiretrovirals. Comorbidities were diversely associated with biomarkers: osteoporosis with higher IL-6, renal impairment with higher sCD14, hypertension with higher D-dimer, diabetes and elevated triglyceride/HDL-cholesterol ratio both with lower adiponectin and lower 25-hydroxyvitamin D. Asymptomatic atherosclerosis and multimorbidity were frequent in a cohort of middle-aged, well-controlled, PLWH and were associated with traditional and HIV-specific factors. Associations between morbidities and inflammatory/immune activation biomarkers were diverse.

Introduction

Combined antiretroviral therapy (cART) has dramatically reduced HIV/AIDS-related mortality and morbidity, allowing to reduce the gap between the life expectancy of people living with HIV (PLWH) and that of the general population.^1,2 However, PLWH experience age-associated comorbidities, in part as a consequence of aging, but with a higher prevalence than matched controls from the general population, and at a younger age, suggesting that these individuals could suffer from accentuated aging and that some personal, HIV-related and/or ART-related factors could be involved.^3
–5 Complications and comorbidities, such as myocardial infarction, stroke, diabetes, dyslipidemia, kidney failure, and osteoporosis, are becoming a major concern in PLWH, with not only a higher prevalence but also a trend toward aggregating in the same individuals.^3,6 Also, persistent residual inflammation in PLWH has been linked to an increased risk of morbidity and mortality, with a significant association with some age-related comorbidities.^6

–10

Inflammation and immune activation play important roles at all steps of atherosclerosis.¹¹ Several biomarkers have been previously associated with cardiovascular risk in PLWH: increased levels of IL-6, C-reactive protein (CRP), and/or D-dimer with myocardial infarction,^12
–14 of sCD14 with progressive or pathological carotid intima-media thickness and calcified coronary plaques,^15,16 and lipoprotein-associated phospholipase A2 (Lp-PLA2) with increased risk of coronary heart disease.^17,18 A low CD4/CD8 T cell ratio has been associated with non-AIDS, particularly cardiovascular, comorbidities.^19,20 Adiponectin has been reported to be negatively associated with insulin resistance, inflammation, and subclinical atherosclerosis in HIV-infected patients.^21
–23 Vitamin D deficiency has also been linked with increased inflammation²⁴ and coronary artery stenosis.²⁵

It is expected that, by 2030, more than 50% of PLWH will have at least two comorbidities.²⁶ Based on these observations, there is an expanding need for clinical screening of major comorbidities among PLWH and for the definition of those most at risk. French guidelines recommend day hospitalization to screen for noncommunicable, non-AIDS comorbidities in PLWH (https://cns.sante.fr/wp-content/uploads/2019/08/experts-vih_comorbidites.pdf). Here, we report a cross-sectional analysis of the cohort of PLWH who attended our multidisciplinary day hospital, examining the prevalence of asymptomatic atherosclerosis, its association with inflammation markers, vitamin D deficiency, and other noncommunicable, non-AIDS comorbidities.

Patients and Methods

Patients

OVHID is an outpatient cohort of PLWH followed at our clinical center. The inclusion in the cohort occurred on the day of admission to the day hospital, a multidisciplinary outpatient facility that allows for same-day comprehensive screening of noncommunicable, non-AIDS comorbidities, according to French guidelines. PLWH referred to the day hospital underwent blood sampling for routine biological tests and biobanking, echocardiography, stress test with or without perfusion myocardial scintigraphy, and carotid artery ultrasound exploration. They also agreed for one blood sample to be frozen and stored for other analysis as well as for their socio-demographic, clinical, and standard-of-care biological data to be used. The OVHID study protocol was approved by the Ethics Committee “Ile de France II” (No ID RCB: 2010-A00417-32) and by the French general data protection regulation (CNIL). All patients gave informed written consent for the use of data and of the biobank.

Imaging procedures

Cardiac and carotid ultrasonographic explorations, stress tests with or without cardiac perfusion scintigraphy were performed according to standard procedures in our hospital. During day hospital, a cardiologist assessed for each patient whether the results should lead to further investigations or treatment. The presence of carotid plaques was assessed by Doppler ultrasonography and the presence of a coronary stenosis was defined as an abnormal stress test, with or without myocardial perfusion scintigraphy, with confirmation by computed tomography angiography or coronary angiogram.

DEXA scan for the assessment of bone mineral density was performed according to standard procedure at our hospital, using a Lunar device.

Biological tests

Routine biological tests were all performed according to standard procedures in the same laboratories of our hospital (Hôpitaux Universitaires Paris Centre). For specific biomarkers, whole blood was recovered with/without EDTA or citrate and plasma/serum was stored at −80° until assayed at the Tenon biochemistry laboratory. The following markers were analyzed as indicated: Lp-PLA2 by immunoturbidimetric assay (PLAC Test; Eurobio, Courtaboeuf, France); inflammatory and immune activation biomarkers, high-sensitivity IL-6 (hsIL-6), sCD14 were analyzed by enzyme-linked immunosorbent assay (ELISA, Quantikine^®; R&D Systems, Abingdon, United Kingdom); high-sentivity C-reactive protein (hsCRP) by immunonephelometry (IMMAGE; Beckman-Coulter, Villepinte, France); D-dimer (plasma) by enzyme-linked fluorescent assay (VIDAS; Biomérieux, Marcy-l'Etoile, France); and Adiponectin was analyzed by ELISA (ALPCO Diagnostics, Salem, NH).

Endpoints

Endpoints were defined a priori as follows. The primary endpoint was the prevalence of a composite criterion assessing the presence of asymptomatic atherosclerotic lesions in the carotid or coronary arteries: presence of carotid plaques (Doppler ultrasonography) or of a coronary stenosis (abnormal stress test, with or without myocardial perfusion scintigraphy, with confirmation by computed tomography angiography or coronary angiogram). Secondary endpoints were (1) the association between asymptomatic atherosclerotic lesions, traditional and HIV-specific risk factors, and the levels of the following biomarkers: hsCRP, IL-6, sCD14, D-dimer, Lp-PLA2, adiponectin, 25-hydroxyvitamin D (25OHD), CD4/CD8 ratio; (2) the association between the levels of markers cited earlier with any comorbidity among cardiovascular diseases (myocardial infarction, carotid plaques, hypertension), renal function impairment, osteoporosis, low-density lipoprotein (LDL)-related and atherogenic dyslipidemia, and diabetes; and (3) the association of multimorbidity (at least two conditions) with the levels of biomarkers.

Renal failure was defined by using the estimated glomerular filtration rate as calculated by the simplified Modification of Diet in Renal Disease formula (<60 mL/min/1.73 m²). Diabetes was defined by fasting glycemia ≥7 mmol/L and/or treatment; LDL-linked dyslipidemia by LDL-cholesterol (LDL-c) ≥4.1 mmol/L and/or treatment with a statin; atherogenic dyslipidemia by a triglyceride (mg/dL)/high-density lipoprotein (HDL)-cholesterol (mg/dL) ratio ≥3.5 for men and 2.5 for women.²⁷ Hypertension was defined as two consecutive measurements of systolic blood pressure of at least 140 mmHg or diastolic blood pressure of at least 90 mmHg, or being on treatment. Osteoporosis was defined as a T-score ≤−2.5 SD at the femoral neck, hip of lumbar spine. Lipodystrophy was defined on the basis of clinical judgment: signs of peripheral lipoatrophy and/or upper trunk or intraabdominal fat accumulation. We used a cut-off of 0.4 for the interpretation of CD4/CD8 ratio as in Trickey et al. ²⁸

Statistical analysis

Choosing the alpha risk at 5% and the power at 80%, we calculated that with a population of around 550 patients with samples available for the measurement of biomarkers and around 200 patients presenting with the primary endpoint, we should be able to show a 1.25 increase of the risk of meeting the primary endpoint for each marker (comparing levels higher vs. lower than the median value in the population), which would be comparable with the literature on HIV-associated cardiovascular disease and, for example, CRP, IL-6, D-Dimers, or fibrinogen levels.²⁹

Associations between the primary endpoint and other items were explored by using Chi-square or Fisher's exact test for categorical variables and Student's or Wilcoxon test for quantitative variables.

A stepwise multivariate logistical model was performed to explore factors associated with the primary endpoint with a significance level of 0.1 required to allow a variable into the model.

Analysis was performed by using SAS 9.1. All tests were bilateral with p < 1%.

Results

Patients' characteristics

Between January 2011 and May 2014, 790 PLWH attended the day hospital and were included in the OVHID cohort. The number of PLW who declined participation was negligible. As shown in Table 1, their median age was 49.8 years (interquartile range [IQR]: 44.5–55.6) and 77% were men. The median time since HIV diagnosis was 15.3 years (IQR: 9.3–20.7), the median nadir CD4 T cell count 211/mm³ (IQR: 90–329), and the median time on cART 9.8 years (IQR: 5.0–13.8). Hepatitis B antigen or hepatitis C antibodies were reported in 8.7% and 14.6%, respectively.

Table 1.

Patients' Characteristics in the Whole Cohort

Variable	Whole cohort n = 790
Age, years	49.8 (44.5–55.6)
Males	77.1
Geographical origin
France	47.3
Subsaharan Africa and others	52.7
Route of HIV acquisition
MSM	46.0
Heterosexual	41.5
IVDU	6.6
Other	5.8
Known duration of HIV infection, in years	15.3 (9.3–20.7)
Current smokers	35.4
Diabetes	19.4
History of cardiovascular event	4.9
Hypertension	33.5
eGFR <60/mL/min	14.1
Osteoporosis	7.9
LDL-c dyslipidemia	13.3
Atherogenic dyslipidemia (TG/HDL ratio)	6.6
Clinical lipodystrophy	18.5
Body mass index	23.7 (21.5–26.4)
Obesity (BMI >30)	9.5
Waist circumference, cm	92 (83–98)
Waist/hip ratio	0.96 (0.92–1.01)
Osteoporosis	7.9
Current CD4 count, cells/mm³	536 (390–754)
CD4 count nadir, cells/mm³	211 (90–329)
CD4 count nadir <200/mm³	46.5
Current CD8 count >800/mm³	39.1
Current CD4/CD8 ratio	0.8 (0.5–1.1)
CD4/CD8 ratio <0.4	14.8
Viral load zenith, log₁₀ RNA copies/mL	4.9 (4.0–5.4)
Viral load currently <40 copies/mL	83.6
Exposure to antiretrovirals, in years	9.8 (5.0–13.8)
No ARV treatment ever	0.5
Ever exposed to PIs	79.2
Ever exposed to NNRTIs	73.9
Ever exposed to d4T	38.6
Cumulative exposure to d4T, years	3.6 (2.1–5.6)
CRP, mg/L (n = 512)	1.6 (0.7–3.9)
IL-6, pg/mL (n = 512)	1.8 (1.2–3.1)
Soluble CD14, pg/mL (n = 512)	1,801.5 (1,536.5–2,142.5)
Lp-PLA2, μg/L (n = 512)	353.3 (270.2–441.9)
D-dimer, ng/mL (n = 512)	341.1 (228.9–531.2)
Adiponectin, mg/L (n = 512)	4.8 (3.3–6.9)
25-OHvitamin D, ng/mL	26 (18–36)
25-OHvitamin D
≥30 ng/mL	42.1
10–30 ng/mL	52.3
<10 ng/mL	5.6

Values are percentage or median value (IQR).

ARV, antiretroviral; BMI, body mass index; CRP, C-reactive protein; d4T, 2′-3-didehydro-2′-3′-dideoxythymidine or stavudine; eGFR, estimated glomerular filtration rate; HDL, high-density lipoprotein; IQR, interquartile range; IVDU, intravenous drug users; LDL-c, low-density lipoprotein cholesterol; Lp-PLA2, lipoprotein-associated phospholipase A2; MSM, men having sex with men; NNRTI, non-nucleosidic reverse transcriptase inhibitor; PI, protease inhibitor; TG, triglycerides.

Because of organizational constraints, inflammation biomarkers were available for 512 individuals out of the 790 participants in the OVHID study.

Characteristics of the patients with available biomarkers were not different from those of the whole cohort (data not shown).

Regarding the inflammation/immune activation biomarkers, the median levels of CRP and IL-6 were 1.6 mg/L and 1.8 pg/mL, respectively, within the normal range. The levels of D-dimers and sCD14 were elevated at 341 ng/mL and 1,801 pg/mL, respectively. These values were comparable to those reported in another French cohort of PLWH, the APROCO-COPILOTE cohort, and higher than those observed in a control group.³⁰

Primary endpoint: atherosclerosis

Out of 790 participants, 204 (26%) were diagnosed with atherosclerosis, in the carotid (presence of carotid plaques at Doppler ultrasonography examination) or coronary arteries (abnormal stress test confirmed by coronarography or computed tomographic angiography).

As shown in Table 2, the 204 individuals with atherosclerosis were older, more frequently men, men having sex with men, and of French origin, had a longer duration of known HIV infection and a longer exposure to antiretrovirals of all classes, and had been more frequently ever exposed to stavudine, had more frequently an undetectable viral load, suffered more frequently from diabetes, hypertension, and clinical lipodystrophy, and had a lower body mass index (BMI) compared with those without atherosclerosis. In addition, the presence of atherosclerosis was significantly and positively associated with having a lower current CD4 count, a CD4/CD8 cell ratio <0.4, higher sCD14 levels, and lower adiponectin levels.

Table 2.

Patients' Characteristics According to the Presence or Absence of Atherosclerosis (Composite Criterion)

Variable	Atherosclerosis n = 204 (26%)	No atherosclerosis n = 586 (74%)	p for comparison
Age, years	53.4 (47.9–59.5)	48.7 (43.2–53.8)	<.0001
Males	84.3	74.6	.004
Geographical origin			.005
France	52.0	45.7
Subsaharan Africa and others	48.0	54.3
Route of HIV acquisition			.04
MSM	52.3	43.8
Heterosexual	33.0	44.5
IVDU	7.6	6.3
Other	7.1	5.4
Known duration of HIV infection, years	18.2 (13.6–22.2)	14.1 (7.5–20.4)	<.0001
Current smokers	39.9	33.8	.1
Diabetes	24.5	17.6	.03
History of cardiovascular event	10.3	3.1	<.0001
Hypertension	43.6	30	.0004
eGFR <60 mL/min	15.5	13.6	.5
LDL-c dyslipidemia	8.8	5.8	.1
Atherogenic dyslipidemia (TG/HDL ratio)	13.7	13.1	.8
Clinical lipodystrophy	25	16.2	.005
Body mass index	23 (21.5–25.5)	24.1 (21.6–26.9)	.005
Obesity (BMI >30)	5.4	10.9	.02
Waist circumference, cm	90 (84–96)	92 (83–98	.3
Waist/hip ratio	0.97 (0.92–1.01)	0.97 (0.92–1.01)	.4
Current CD4 count, cells/mm³	537 (403–754)	558 (358–694)	.0002
CD4 count nadir, cells/mm³	196 (75–329)	220 (100–331)	.3
CD4 count nadir <200/mm³	50.5	45.1	.2
Current CD8 count >800/mm³	42.8	37.8	.07
Current CD4/CD8 ratio	0.8 (0.6–1.1)	0.8 (0.5–1.1)	.3
CD4/CD8 ratio <0.4	16.4	10	.03
Viral load zenith, log₁₀ RNA copies/mL	4.8 (4.0–5.4)	4.9 (4.0–5.4)	.8
Viral load currently <40 copies/mL	89.2	81.7	.02
Exposure to antiretrovirals, in years	12.2 (6.9–15.3)	8.9 (4;4–13.1)	<.0001
No ARV treatment ever	1.0	0.4	.3
Ever exposed to PIs	83.8	77.5	.06
Ever exposed to NNRTIs	79.4	71.9	.04
Ever exposed to d4T	49.5	34.5	<.001
Cumulative exposure to d4T, years	3.7 (2.1–5.6)	3.6 (2.1–5.3)	.6
CRP, mg/L (n = 512)	1.5 (0.7–3.1)	1.7 (0.7–4.1)	.2
IL-6, pg/mL (n = 512)	1.8 (1.2–3.0)	1.8 (1.2–3.2)	.7
Soluble CD14, pg/mL (n = 512)	1,877 (641–2,286)	1,778 (491–2,110)	.0008
Lp-PLA2, μg/L (n = 512)	356.4 (290.1–426.4)	347.5 (262.2–446.5)	.7
D-dimer, ng/mL (n = 512)	346.7 (222.9–547.0)	339.2 (228.9–526.0)	.9
Adiponectin, mg/L (n = 512)	4.4 (3.0–6.2)	5 (3.6–7.1)	.006
25-OHvitamin D, ng/mL	25 (17–36)	27 (18–36)	.3
25-OHvitamin D			.2
≥30 ng/mL	37.0	43.8
10–30 ng/mL	55.6	51.2
<10 ng/mL	7.4	5.0

Values are percentage or median value (IQR).

There was no significant difference for HBV or HCV status, renal impairment prevalence, or 25OHD levels.

In multivariate analysis (Table 3), older age [per 10-year increase; odds ratio (OR): 1.6, 95% confidence interval (CI): 1.2–2.1, p = .003], longer duration of known HIV infection (per 10-year increase; OR: 1.1, 95% CI: 1.0–1.1, p < .0001), and sCD14 levels (per 500 pg/mL increase; OR: 1.3, 95% CI 1.0–1.7, p = .02) remained significantly and positively associated with the presence of atherosclerosis; whereas higher BMI (per 1 unit increase; OR: 0.9, 95% CI: 0.9–1.0), detectable viral load (yes vs. no; OR: 0.4, 95% CI 0.2–0.9, p = .03), and adiponectin levels (per 1 mg/L increase; OR: 0.9, 95% CI 0.9–1.0, p = .02) were negatively associated.

Table 3.

Multivariate Analysis of Factors Associated with the Presence of Atherosclerosis (Composite Criterion)

Variable	Univariate regression		Multivariate model
Variable	OR (95% CI)	p	Adjusted OR (95% CI)	p
Age, per 10-year increase	1.8 (1.5–2.2)	<.0001	1.6 (1.2–2.1)	.003
Known duration of HIV infection, per 10-year increase	1.9 (1.5–2.4)	<.0001	2.1 (1.5–3.0)	<.0001
Body mass index, per unit	0.9 (0.9–1.0)	.005	0.9 (0.9–1.0)	.003
Viral load >40 copies/mL, yes vs. no	0.5 (0.3–0.9)	.02	0.4 (0.2–0.9)	.03
sCD14, per 500 pg/mL increase	1.4 (1.1–1.7)	.0008	1.3 (1.0–1.7)	.02
Adiponectin, per 1 mg/L increase	0.9 (0.8–1.0)	.006	0.9 (0.9–1.0)	.02

CI, confidence interval; OR, odds ratio.

Only 25 participants, all males, had proven coronary artery stenosis. Compared with those without documented coronary disease, they were significantly older, had a longer known duration of HIV infection and treatment, and presented more often with a personal history of cardiovascular events and hypertension. They tended to have higher sCD14 levels, but this did not reach statistical significance. In the multivariate analysis, only a personal history of cardiovascular events remained associated with coronary stenosis (adjusted OR: 25.5, 95% CI: 13.4–135.3, p = .002).

Secondary endpoint: other comorbidities

Hypertension and diabetes were found in 33.5% and 19.4% of participants, respectively, renal function impairment in 14.1%. A history of cardiovascular disease was reported by 4.9% of participants. LDL-related dyslipidemia was present in 13.3% of individuals, whereas an abnormal triglyceride/HDL-cholesterol ratio was reported in 6.6%. Osteoporosis was found in 7.9% of participants. Regarding tobacco use, 35.4% of participants were active smokers.

We also assessed the percentage of patients with multimorbidity, defined as at least two comorbidities among: atheroma (composite criterion), hypertension, abnormal triglycerides (TG)/HDL-cholesterol ratio, LDL-related dyslipidemia, diabetes, renal impairment, and osteoporosis. The distribution of participants with regard to comorbidities was as follows: 24.7% with none, 33.2% with one, 24.7% with two, and 17.4% with three or more.

Hypertension was significantly associated with older age (52.6 vs. 48.5 years, p = .0001), diabetes (29.4% vs. 14.3%, p = .006), history of cardiovascular disease (12.1% vs. 1.3%, p = .0001), renal dysfunction (26.3% vs. 8.1%, p = .0001), high LDL-c (17.7% vs. 11.0%, p = .009), higher BMI (24.6 vs. 23.3, p = .0001), higher waist circumference (94 vs. 90 cm, p = .002), lower CD4 T cell count nadir (164 vs. 232/mm³, p = .0006), longer exposure to ART (10.3 vs. 9.3 years, p = .03), more frequent exposure to stavudine (47.1% vs. 34.3%, p = .0001), and higher D-dimer levels (394.9 vs. 314.6 ng/mL, p = .001). It was also associated with less frequent smoking (28.6% vs. 38.7%, p = .006).

Diabetes was significantly associated with older age (52.2 vs. 49.1 years, p < .0001), less tobacco smoking (28.6% vs. 37.4%, p = .01), history of cardiovascular disease (9.1% vs. 3.9%, p = .01), hypertension (51.0% vs. 29.4%, p = .0001), longer exposure to ART (11.8 vs. 9.3 years, p = .001), exposure to stavudine (47.7% vs. 36.4%, p = .01), lipodystrophy (25.5% vs. 16.8%, p = .01), higher BMI (24.4 vs. 23.5, p = .001), waist circumference (94 vs. 91 cm, p = .009), and waist-to-hip ratio (0.99 vs. 0.96, p = .0002), and lower 25OHD levels (Table 3).

Renal dysfunction was significantly associated with older age (53.3 vs. 49.0 years, p < .0001), hypertension (61.4% vs. 28.2%, p = .0001), lower current CD4 T cell count (492 vs. 558/mm³, p < .0001) and CD4 nadir (158 vs. 222/mm³, p = .0001), as well as higher sCD14 (1970 vs.1792 pg/mL, p = .002).

LDL-c dyslipidemia was significantly associated with hypertension (44.8% vs. 31.8%, p = .01), clinical lipodystrophy (25.5% vs. 16.8%, p = .01), and higher current CD4 count (601 vs. 541/mm³, p < .0001).

Abnormal TG/HDL-cholesterol ratio was significantly associated with male gender (90.4 vs. 76.1, p = .01), history of cardiovascular disease (11.5% vs. 4.5%, p = .01), higher current CD4 (591 vs. 549/mm³, p < .001), exposure to protease inhibitors (94.1% vs. 78.1%), lower adiponectin (3.1 vs. 4.8 mg/L, p = .0006), and lower 25OHD (Table 3).

Osteoporosis was significantly associated with smoking (50.9% vs. 33.3%, p = .01), lower BMI (21.1 vs. 24.0, p = .001), exposure to stavudine (49.1% vs. 61.5%, p = .01), and higher IL-6 levels (2.9 vs. 1.8 pg/mL, p = .01).

No association was found between HBV or HCV infection and any of these comorbidities.

Biomarkers were differently associated with comorbidities: higher IL-6 with osteoporosis, higher sCD14 with renal impairment, higher D-dimer with hypertension, lower adiponection and lower 25OHD with diabetes, and high TG/HDL. No significant association with the studied markers was found for LDL-c.

Multimorbidity (at least two comorbidities) was present in 42% of the participants and was significantly associated with older age, longer duration of known HIV infection, history of cardiovascular disease, higher waist circumference and higher waist-to-hip ratio, lower current CD4, longer exposure to protease inhibitors and ever exposure to stavudine, and higher levels of sCD14. In multivariate analysis, only older age (per 10-year increase: OR 2.1, 95% CI: 1.6–2.7, p < .0001) and longer exposure to cART (per 10-year increase: OR 1.05, 95% CI, 1.01–1.09, p = .004) remained significantly associated with multimorbidity.

Discussion

We studied the prevalence of atherosclerosis and associated comorbidities in a large cohort of PLWH who had been referred to a day hospital for comprehensive screening of non-AIDS noncommunicable diseases.

Regarding our primary endpoint, we show here that 26% of participants were diagnosed with silent atherosclerosis, defined by the presence of carotid plaques (Doppler ultrasonography) or of a coronary stenosis (confirmed by CT angiography or coronary angiography). Multivariate analysis showed a positive association with age, known duration of HIV infection, lower BMI, undetectable viral load, higher sCD14, and lower adiponectin.

Patients were asymptomatic with regards to atherosclerosis, which stresses the need for screening, especially if traditional risk factors are reported. Indeed, atherosclerosis was significantly associated with age, diabetes, and hypertension. Participants with atherosclerosis were more frequently active smokers, but this did not reach statistical significance. This may be due to the fact that we could not clearly distinguish between patients with recent or prolonged smoking cessation, or to the weight of HIV-related factors in this population with a median duration of HIV infection of more than 15 years. HIV-related factors associated with atherosclerosis were a longer known duration of HIV carriage, a longer exposure to cART, and more frequent exposure to stavudine, reflecting exposure to first-generation, toxic antiretroviral drugs. The long-lasting role of stavudine, even stopped for years, in the occurrence of remaining fat redistribution and associated hypertension and dyslipidemia has been recently recognized.^31,32 Atherosclerosis was also associated with having an undetectable viral load. This may seem counterintuitive since studies have shown a positive association between ongoing HIV replication and myocardial infarction⁸; however, this could also reflect a better adherence to antiretroviral treatment, the exposure to which, appearing in itself, is a risk factor.

We found that a lower BMI was associated with atherosclerosis. This is in keeping with data from the D:A:D cohort study³³ and might be due to the exposure to first-generation drugs such as stavudine, responsible for lipoatrophy and persistent decreased body weight.^31,34,35

Higher sCD14 concentrations were significantly associated with atherosclerosis in the multivariate analysis, in keeping with previous studies,^36,37 stressing the role of innate immunity activation in vascular alterations. However, although it can be useful at the population level, the interpretation of sCD14 levels on an individual basis remains unclear.³⁸ Indeed, in a systematic review performed in 2017, Vos et al. did find frequent associations between inflammatory biomarkers and a variety of cardiovascular surrogate markers across studies in PLWH, but they could not identify any constant association pattern.³⁵

In our study, the presence of atherosclerosis was also associated with a lower CD4/CD8 ratio, which has been proposed, in PLWH on cART, as an integrative marker of persistently altered T cell populations balance, innate and adaptive immunity activation, associated in some studies^20,39,40 with poorer clinical outcomes, although this has been challenged by others.²⁸ We chose a cut-off of 0.4, as in Trickey et al.,²⁸ to examine the association between the CD4/CD8 ratio and atherosclerosis, but Mussini et al. showed that a ratio <0.3 was associated with non-AIDS comorbidities in patients on suppressive cART,⁴⁰ whereas Castilho et al. ²⁰ showed a protective effect of every 0.1 increment of CD4/CD8 ratio on the risk of coronary disease, without identifying a definite cut-off. Since the CD4/CD8 ratio is easily available, we believe that a very low value can be helpful for the characterization of patients' risk.

Lower levels of adiponectin were associated with the presence of atherosclerosis, in keeping with previous reports in PLWH examining either carotid intima-media thickness,²² its progression over time,⁴¹ or the presence of coronary artery stenosis²³ Adiponectin favors insulin sensitivity and lower levels have been associated with HIV treatment-related lipodystrophy,⁴² which is coherent with these findings. Lp-PLA2, described as associated with cardiovascular risk^17,18 was not associated with atherosclerosis or hypertension in the current study.

Besides atherosclerosis, our population presented with a variety of age-related comorbidities. Even if their prevalence varied among the different studies performed in quite similar patients, globally they were high^3,6,43 and higher than in a matched HIV-negative population of a similar age.³ In the current study, besides age, factors associated with hypertension, diabetes, renal dysfunction, dyslipidemia, and osteoporosis were as expected in the general population. In addition, exposure to first-generation treatment of HIV infection, in particular stavudine, was frequently associated with these conditions.

Multimorbidity was present in 42% of the participants, which is high, but difficult to compare with other cohorts since the comorbidities considered are not the same. Multimorbidity was significantly associated with personal history (age, history of cardiovascular disease), anthropometric measurements (higher waist circumference and higher waist-to-hip ratio), factors related to HIV infection history (longer duration of known HIV infection, lower current CD4), and longer exposure to antiretroviral drugs (particularly protease inhibitors, and ever exposure to stavudine), together with higher sCD14 levels. In the AGEhIV cohort, most of these factors were similarly associated with multimorbidity,³ further demonstrating the multifactorial origin of this status.

It should be emphasized that the levels of some of the inflammatory biomarkers were, indeed, associated with certain comorbidities (IL-6 with osteoporosis, D-dimer with hypertension, sCD14 with atherosclerosis, and renal impairment) or multimorbidity (sCD14), but the associations differed among the different comorbidities that were examined. A similar observation was made in the APROCO-COPILOTE study.⁶ This suggests that the pro-inflammatory status observed in these patients involves different mechanisms according to the comorbidity considered. At present, there is no indication that a few selected biomarkers can globally help in evaluating the pro-inflammatory status of PLWH at the individual level.

The strengths of our study are that endpoints, including atherosclerosis, were documented through standardized diagnostic procedures, that patients were homogeneously managed in a single center, with investigations performed in the same hospital departments, with the same equipment. Also, the 790 individuals enrolled in our study had similar sociodemographic, clinical, and biological characteristics than PLWH enrolled in the large French Hospital database on HIV,⁴⁴ or in the AQUITAINE outpatient cohort of PLWH,⁴³ which makes it representative of the current French population of PLWH.

However, our study has several limitations. First, we cannot rule out a bias in participants' selection, due to the design of our study, based on routine clinical care: patients with risk factors may have been more likely to be referred to the day-care hospital than those without. Second, the clinical significance of asymptomatic atherosclerosis is unclear in terms of individual prediction of cardiovascular events.⁴⁵ Also, we only had limited access to information that could help interpreting some of the results, such as former smoker status (which could explain why patients with hypertension and diabetes were less frequently active smokers) and prescription of/adhesion to vitamin D supplementation (which could explain the absence of difference in 25OHD levels in patients with and without osteoporosis). Finally, the absence of an HIV-negative control group precludes comparisons with the general population.

Conclusion

To conclude, asymptomatic atherosclerosis and multimorbidity were frequent in a cohort of middle-aged, well-controlled PLWH, and were associated with traditional risk factors (above all, age) and HIV-specific items (principally durations of HIV carriage and treatment). Associations with biomarkers were diverse among the conditions that we examined. Altogether, these data underline the need for active monitoring and treatment of cardiovascular risk factors.

Footnotes

Acknowledgments

The authors wish to thank all participants, physicians who referred patients for investigations, especially Dr. Linda Belarbi, Dr. Jacques Gilquin, Dr. Blanca Hadacek, Dr. Leila Naït-Ighil, Dr. Thu-Huyen Nguyen, Dr. Olivier Zak-dit-Zbar, and the staff at the clinical research center, in particular Ms. Sandra Colas and Prof. Jean-Marc Tréluyer, for monitoring and support.

Authorship Statement

J.G., H.A., A.M., and J.-P.V. designed the study. J.G., A.M., D.S., J.-P.M., J.D., and J.-P.V. contributed to the definition of protocols and referred patients for investigations. J.B. supervised cardiologic investigations and analyses. S.F., J.-P.B., and J.C. designed and performed biological analyses. H.A. performed the statistical analyses. All authors contributed significantly to the drafting, revising, and final approval of the article.

All authors account for all aspects of the submitted work.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was funded by SIDACTION-Ensemble Contre le Sida (AO26-1).

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