Assessment of cardiac diastolic dysfunction in HIV-infected people without cardiovascular symptoms in China

Abstract

This study assessed cardiac diastolic dysfunction (DD) in HIV-infected subjects without cardiovascular symptoms in China. Eighty-four HIV-infected patients without cardiovascular symptoms were recruited and compared with 30 HIV-negative control subjects. Cardiac DD was evaluated using echocardiography including tissue Doppler imaging. DD was detected in 47.6% of AIDS patients and 42.8% of HIV-positive patients, which was significantly higher than in HIV-negative controls. After logistic regression analysis, HIV infection was independently correlated with cardiac DD (P < 0.05). Among HIV-positive subjects, logistic regression analysis showed that zidovudine exposure was associated with higher prevalence of DD (P < 0.05). Asymptomatic cardiac DD was observed frequently in HIV-infected subjects. HIV infection itself and zidovudine exposure were correlated with a higher prevalence of cardiac DD.

Keywords

HIV HAART echocardiography zidovudine diastolic dysfunction

INTRODUCTION

There is a growing recognition of increased cardiovascular risk among HIV-infected patients. HIV infection is associated with increased rates of cardiovascular events.^1,2 The higher prevalence of risk factors such as hyperlipidaemia and insulin resistance related to highly active antiretroviral therapy (HAART) has further increased the cardiovascular risk.^3–5 Studies of HIV-negative patients without cardiac symptoms show that diastolic dysfunction (DD) is an early marker of underlying cardiac disease.^6,7 To investigate this concern, we evaluated cardiac DD in HIV-infected subjects.

The aims of the present study were: one, to compare the prevalence of cardiac DD between AIDS patients who have received HAART, HIV-positive ‘carriers’ who had never received antiretroviral therapy (ART) and HIV-negative controls and, two, to study the association between cardiac DD with HIV-related factors and cardiovascular risk factors.

SUBJECTS AND METHODS

Study population

Eighty-four ambulatory HIV-infected subjects aged 30–50 years were recruited to this study through the Infectious Diseases Clinic of the Peking Union Medical College (PUMC) hospital between June 2007 and October 2008. Forty-two patients had clinical AIDS according to the World Health Organization (WHO), definition and all these patients had received HAART for more than 12 months, including two nucleoside reverse transcriptase inhibitors (NRTIs) as backbone and either a non-nucleoside reverse transcriptase inhibitors (NNRTIs; nevirapine or efavirenz) or a protease inhibitor (PI; indinavir or lopinavir/ritonavir). The AIDS subjects had a mean CD4 count of was 394 cells/μL (range 32–705) with a mean CD4 nadir of 114 cells/μL (range 4–290). Forty-two HIV-positive ‘carriers’ were naïve to ART and were recruited from a clinical study of long-term non-progressors in the Infectious Diseases Clinic of the PUMC hospital. Their mean CD4 count was 444 cells/μL (range 336–1016) and the mean possible HIV infection duration was 14 years. Thirty HIV-negative controls were recruited during the same period, matched with HIV-positive subjects for age, sex and proportion with hypertension and diabetes. Patients and controls underwent uniform clinical evaluation and the following data were recorded for each subject: age, sex, weight, height, blood pressure at rest, opportunistic infections, ART history, presence of history of hypertension, diabetes, smoking and current symptoms of cardiovascular disease. Subjects with documented history/symptoms attributable to cardiovascular disease and/or an abnormal 12-lead electrocardiogram were excluded from the study. Written informed consent was obtained from each subject.

Laboratory measurements

Blood was drawn from each subject after an overnight fast on the morning of the study visit. Total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined by enzymatic methods using commercial kits. CD4+ and CD8+ lymphocyte cell counts were analysed by three-colour flow cytometry (Epics XL flow cytometry, Beckman Coulter, Miami, FL, USA). Whole blood was incubated with monoclonal antibodies against CD3, CD4, CD8, CD38 and DR (Immunotech, Marseilles, France). Absolute counts of CD3 + CD4+ cells, CD3 + CD8+ cells were then calculated using the results from the complete blood cell and lymphocyte differential counts. HIV-1 viral load (VL) was measured on frozen ethylenediaminetetraacetic acid-plasma samples by a branched DNA assay (version 3.0, Bayer, Tarrytown, NY, USA) with a limit of detection from 50 copies/mL to 50,000 copies/mL.

Echocardiographic measurements

Transthoracic echocardiography was performed and interpreted by the same cardiologist, masked to the HIV status of the subjects. All studies were performed on a General Electric Vivid 7 ultrasonograph (GE Healthcare, Milwaukee, WI, USA). Two-dimensional measurements were performed according to recommendations of the American Society of Echocardiography. Measurement of ejection fraction was performed by quantitative 2-D (biplane Simpson) method. Pulsed Doppler was used to record transmitral flow in the apical four-chamber view. Tissue Doppler velocities were then acquired at the septal and lateral annular sites. Mitral inflow measurements included peak early (E) and peak late (A) velocities, the E/A ratio and E wave deceleration time (EDT). Pulsed Doppler was also used to record transmitral flow and left-ventricular outflow flow in the apical five-chamber view and to measure left-ventricular isovolumic relaxation time. Systolic, early diastolic and late diastolic velocities by tissue Doppler at the septal and lateral annular sites were measured. Using the average of septal and lateral early diastolic velocities (Ea), the E/Ea ratio was computed.⁸ Three cardiac cycles were measured and averaged for all Doppler measurements.

Diastolic function was categorized according to the progression of DD as follows: normal; mild impaired relaxation subjects who had mitral inflow patterns with reduced E/A ratios (<1.0) and EDT >250 ms; moderate impaired relaxation (pseudonormal) subjects with increased E/A ratios (>1.0), increased E/Ea ratios (>10) and EDT that was not reduced (>150 ms); and severe impaired relaxation (restrictive) subjects with increased E/A ratios (>1.5), increased E/Ea ratios (>10) and reduced EDT (<150 ms).^6,9

Statistical analysis

Statistical analysis was performed using SPSS 11.5 (SPSS Inc, Chicago, IL, USA). Continuous variables were presented as means and SDs or medians. Viral load data were logarithmically transformed to obtain near normality before analysis. Because some data were not normally distributed, as determined with the Kolmogorov–Smirnov test, Wilcoxon rank sum was used to compare variables. A χ ² test was used to compare categorical variables. Multiple linear regressions models, using the forced entry method, were constructed to determine statistically significant independent variables that explain variation in cardiac diastolic function. To investigate the impact of HIV-related factors and antiretroviral medication exposure on the prevalence of DD, logistic regression was performed. A P value of 0.05 was used to test for statistical significance, and all statistical tests were two tailed.

RESULTS

Clinical and biologic characteristics

Clinical characteristics of the study populations and control group are listed in Table 1. The mean age, gender, hypertension and diabetes mellitus percentages were not significantly different between the three groups. AIDS patients showed lower mean weight and body mass index, probably related to their long pre-HAART disease history as well as malnutrition. The median current CD4 and CD8 counts of the AIDS and HIV-positive carriers groups were not significantly different. In the AIDS group, the mean duration of HAART was 32 months. At inclusion, all patients were using two NRTIs as backbone of their HAART. These included zidovudine (27 patients), lamivudine (33 patients), stavudine (10 patients) and didanosine (14 patients). Most of the AIDS patients were treated with nevirapine (NVP) and efavirenz (EFV) as their NNRTI (NVP 24 patients, EFV 12 patients) and six patients (14.3%) were treated with two NRTIs and a PI.

Table 1

Baseline characteristics of the study group and control group

	AIDS patients (n=42)	HIV+carriers (n=42)	Controls (n=30)	P value
Anthropometric parameters
Age (years)	40.6±9.3	38.6±6.5	41.5±10.5	NS
Male/female (n)	25/17	25/17	18/12	NS
Height (m)	1.65±0.09	1.62±0.08	1.66±0.07	P<0.05*
Weight (kg)	58.54±7.66	67.27±10.68	63.62±10.23	P<0.01^†
Weight (kg)				P<0.05^‡
BMI (kg/m²)	21.85±2.54	25.48±3.46	22.91±2.93	P<0.01^†
BMI (kg/m²)				P<0.01*
Cardiac vascular risk factors
Systolic BP at rest (mmHg)	127±13	131±14	127±22	NS
Diastolic BP at rest (mmHg)	76±11	77±9	79±14	NS
Tobacco use (n[%])	18 (42.9)	21 (50)	16 (53.3)	P<0.05*
Hypertension (n [%])	7 (16.6)	9 (21.4)	7 (23.3)	NS
Diabetes (n[%])	4 (9.5)	3 (7.2)	3 (10)	NS
Cholesterol (mmol/L)	3.95±0.78	3.83±0.82	4.24±0.86	P<0.05*
HDL-C (mmol/L)	1.21±0.42	1.11±0.26	1.15±0.34	NS
LDL-C (mmol/L)	2.04±0.42	2.15±0.65	2.53±0.34	P<0.05*
LDL-C (mmol/L)				P<0.05^‡
Triglycerides (mmol/L)	1.31 (0.52–11.62)	1.18 (0.55–3.32)	1.09 (0.54–2.56)	NS
Glucose (mmol/L)	4.38±1.88	3.73±1.05	4.80±0.95	P<0.05*
Lipid lowering medication use (n)	7	0	3	P<0.05^†
HIV/AIDS-related factors
Duration of HIV seropositivity (Mo)	158.4±24.8	168.8±22.0	ND	NS
Current CD4 count (cells/μL)	394 (32–705)	444 (361–1046)	ND	NS
Nadir CD4 count (cells/μL)	114 (4–290)	430 (336–1016)	ND	P<0.001^†
VL(log copies/mL)	2.08±0.79	3.34±0.99	ND	P<0.001^†
Current CD8 count (cells/μL)	695±264	694±339	ND	NS
CD8+DR+ %	23.5±11.7	39.5±12.3	ND	P<0.001^†
CD8+CD38+ %	40.5±19.5	59.4±15.7	ND	P<0.001^†

BMI = body mass index; BP = blood pressure; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; ND = not determined; NS = not significant

*HIV+ carriers versus HIV−controls

^†AIDS versus HIV+ carriers

^‡AIDS versus HIV− controls

Values are means ± SD or median (interquartile range)

Echocardiographic measurements

Using the defined echocardiographic criteria, six subjects in this study could not be classified into any of the groups, and hence their Ea values will be referred to. If their Ea value was less than 8 cm/second, they were recognized as having DD.^10,11 E/Ea values <10 can also guide us to distinguish normal one from pseudonormal DD. Of the 84 HIV-infected subjects enrolled, 38 subjects (45.2%) showed evidence of DD by the defined echocardiographic criteria. Mild impaired DD was detected in 13 AIDS patients and nine HIV-positive carriers and moderate impaired DD was found in five AIDS patients and eight HIV-positive carriers. Only two AIDS patients and one HIV-positive carrier had severe DD. The prevalence of DD in the AIDS and HIV-positive carrier groups were both significantly higher than the control group (P < 0.05).

Parameters of DD are listed in Table 2. The conventional diastolic parameter E/A ratios were not significantly different between AIDS patients, HIV-positive carriers and the control group. Using a more comprehensive evaluation method, significant differences in diastolic variables were discovered. Significant differences were found between the HIV-positive carriers and the control group (EDT 166.28 versus 190.34, P < 0.05). Significant differences were also detected between the AIDS group and the control group (EDT 161.87 versus 190.34, P < 0.05).

Table 2

Left-ventricular diastolic function characteristics of the study samples

Variables	AIDS patients (n = 42)	HIV+ carriers (n = 42)	Controls (n = 30)	P value
E/A ratio	1.16 ± 0.35	1.20 ± 0.40	1.19 ± 0.26	NS
E/Ea ratio	5.43 ± 1.99	5.68 ± 1.70	5.78 ± 0.91	NS
E wave deceleration time (ms)	161.87 ± 21.64	166.28 ± 20.26	190.34 ± 37.22	P < 0.05*
E wave deceleration time (ms)				P < 0.05^†
Left-ventricular isovolumic relaxation time (ms)	93.18 ± 20.34	103.48 ± 18.35	93.57 ± 18.55	P < 0.05^‡
Ea (cm/s)	10.18 ± 2.80	10.42 ± 2.95	11.45 ± 2.75	NS
Diastolic dysfunction (n)	20	18	6	P < 0.05*
Diastolic dysfunction (n)				P < 0.05^†

E/A ratio = peak E wave velocity/ peak A wave velocity; Ea = the average of septal and lateral systolic early diastolic velocity by tissue Doppler; NS = not significant

*HIV+ carriers versus HIV− controls

^†AIDS versus HIV − controls

^‡AIDS versus HIV+ carriers

Relationship of DD to cardiovascular risk factors and HIV-related factors

As shown in Table 3, in the AIDS patients, HIV-positive carriers and control group, logistic regression analysis shows that HIV infection is an independent risk factor for DD (P < 0.05). However, traditional cardiovascular risk factors such as blood pressure, diabetes and LDL-C did not show independent correlation with DD.

Table 3

Logistic regression analysis of factors of diastolic dysfunction in HIV+ and HIV− subjects

	OR	95% CI	P value
HIV infection	17.00	3.33–86.9	0.001*
HAART	0.46	0.09–2.44	0.36
Age (years)	1.00	0.93–1.07	0.98
Sex	1.17	0.27–5.09	0.80
Body mass index (kg/m²)	0.90	0.73–1.10	0.30
Tobacco use (n)	1.32	0.29–6.07	0.72
Diabetes (n)	0.22	0.03–1.34	0.10
Hypertension (n)	0.841	0.25–2.85	0.78
LDL-C > 130 mg/dL (n)	2.28	0.47–10.94	0.30

CI = confidence interval; HAART = highly active antiretroviral therapy; LDL-C = low-density lipoprotein cholesterol; OR = odds ratio

*P < 0.05

Dyslipidemia was defined as serum triglyceride is more than 1.7 mmol/L (150 mg/dL) or serum HDL-C is less than 0.91 mmol/L (35 mg/dL) or serum LDL-C is more than 3.36 mmol/L (130 mg/dL).^12,13 As shown in Table 4, among the AIDS subjects, logistic regression analysis revealed that zidovudine exposure was independently associated with cardiac DD (P < 0.05). The median viral load, nadir CD4 count and current CD8 count were not independent risk factors. In addition, the traditional cardiovascular-related factors such as BMI, diabetes, dyslipidemia did not show independent correlation with DD in AIDS patients.

Table 4

Logistic regression analysis of factors of diastolic dysfunction in AIDS patients

	OR	95% C.I.	P value
Sex	16.8	0.86–328.31	0.063
Age (years)	0.991	0.879–1.119	0.885
Body mass index (kg/m²)	0.765	0.46–1.272	0.302
Tobacco use (n)	7.647	0.427–136.83	0.167
dyslipidemia (n)	1.019	0.113–9.18	0.986
CD4 cell count <200 (n)	0.575	0.082–4.06	0.579
CD8 cell (cells/μL)	1.003	0.998–1.01	0.208
Viral load (log copies/mL)	1.017	0.245–4.21	0.982
AZT use (n)	25.416	1.052–614.32	0.046*
D4T use (n)	3.39	0.106–108.71	0.49

AZT = zidovudine; CI = confidence interval; d4T, stavudine; OR = odds ratio

*P < 0.05

DISCUSSION

In this cross-sectional study of HIV-infected subjects without symptoms of cardiovascular disease, we observed 47.6% of AIDS patients and 42.8% of HIV-positive carriers had DD, most of which was mild or moderate. In the study by Nayak et al. ¹⁴, the prevalence of DD in a cohort of HIV-infected patients on HAART at low risk for AIDS and cardiovascular disease was 37%.

Before the ART era, investigators described a high incidence of DD in HIV-infected patients.^15,16 Very few studies screened for silent cardiac abnormalities in AIDS patients with well-controlled viral load since the introduction of combination ART.¹⁷ Our study screened both HIV-positive carriers not on ART and AIDS patients receiving HAART for more than 12 months without symptoms of cardiovascular disease. Unlike previous studies using conventional echocardiography in the HIV-infected population, we were able to perform a more in-depth evaluation of diastolic function integrating conventional and novel diastolic parameters, including tissue Doppler imaging.^18,19 The conventional evaluation of diastolic function is highly dependent on loading conditions, and in the absence of additional parameters, it is impossible to distinguish between the normal and the pseudonormal transmitral flow pattern. Therefore, the conventional evaluation method may underestimate the prevalence of DD. Schuster et al. ²⁰ reported that using a multiparametric approach for the evaluation of diastolic function in HIV-positive patients with E/A ratio > 1, they diagnosed the pseudonormal filling pattern (moderate impaired DD) in an additional 37% of patients. Our study is also the first study examining cardiac diastolic function of Chinese HIV-infected subjects.

Our results showed that HIV-infected subjects had higher prevalence of DD than HIV-negative controls. HIV infection is an independent risk factor for DD, which suggests a possible direct effect of HIV on the myocardium. Multiple factors may be involved in the increased prevalence of DD in the setting of HIV infection. Animal and autopsy studies indicated that HIV virions affected cardiomyocytes directly and were associated with local release of cytokines leading to inflammation, myocarditis and dilated cardiomyopathy.^21–23

A potentially important finding is that using zidovudine for more than 12 months showed a trend towards increased cardiac DD. Mitochondrial toxicity of zidovudine, largely described in various tissues, including cardiomyocytes, is a worrying concern. In animal models and in humans, there is evidence to suggest that cardiomyopathy is caused by the use of zidovudine.^24–29

Prior studies have shown that there was a direct relationship between the cardiovascular risk factors and DD but our results did not confirm these findings. This might be explained by our study population's generally low cardiovascular risk and the low percentage of subjects with hypertension, diabetes and other cardiovascular risk factors.

The study by Meng et al. ¹⁷ had shown that continued PI exposure was correlated with increased DD. However, our study did not indicate that PI exposure was associated with higher prevalence of DD. The disparate result may be related to the few patients who used PIs in our study (only 6 patients). An international collaborative study reported that patients exposed to abacavir and didanosine had a greater risk of coronary heart disease than those prescribed other NRTIs (D:A:D study).³⁰ Review of our patients with exposure to didanosine did not show a higher prevalence of DD.

DD of the heart is a complex sequence of multiple interrelated events, which has been difficult to evaluate. There are many measures and indices to assess and the diagnosis criteria for DD are not unified. We used a precise criterion that has been widely accepted in the routine clinical setting. The limitation of this criterion is that it might not detect all subjects. In our study, there were six subjects who were unable to be classified into any of the groups. Peak early diastolic mitral velocity (Ea) is relatively load independent and correlates to isovolumic relaxation. An Ea value of less than 8 cm/s is consistent with DD¹⁰ and the Ea value helped us to categorize DD in these few subjects.

There were several limitations to our study. The small sample size resulted in a decreased power to detect statistically significant differences. Cardiac abnormalities might result from complex interactions between HIV infection itself, ART medication and other HIV-unrelated factors such as smoking, obesity, hypertension and diabetes. Studies on larger cohorts of strictly selected patients will be needed to evaluate the multifactorial pathogenesis of DD in HAART-experienced patients. In addition, this study is cross-sectional and future follow-up of this cohort for cardiac structural changes and clinical outcomes is warranted. Prospective studies should be pursued to further define risk factors in HIV-infected subjects.

Our study demonstrated that in an HIV-infected population at low cardiovascular risk without cardiovascular symptoms, DD had a surprisingly high prevalence when compared with the general population of similar age and was possibly influenced by a combination of HIV infection itself and exposure to zidovudine.

Footnotes

ACKNOWLEDGEMENTS

This study was supported by National Key Technologies R&D Program for the 11th Five-year Plan (2008ZX10001-006), Ministry of Health Clinical HIV/AIDS Research Grant(2007–2009) and Beijing Science and Technology Program Fund (D0906003040491).

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