Retinal Vascular Parameter Variations in Patients With Human Immunodeficiency Virus

Abstract

Purpose.: To compare the retinal vascular parameters in patients with human immunodeficiency virus (HIV) infection with normal controls, and to determine the relationship between retinal vascular parameters and HIV-related blood biomarkers (CD4⁺ T-lymphocytes count, presence of HIV RNA).

Methods.: Case-control study of eighty-five patients with HIV on follow-up at the Communicable Disease Center, Singapore, and 251 age-, sex-, and race-matched normal healthy controls (case: control matching ≈ 1:3) selected from the Singapore Epidemiology of Eye Disease program were included in this study. Standardized retinal photographs were taken from patients and controls. Trained technicians measured quantitative retinal vascular parameters (retinal vascular caliber, branching angle, tortuosity, and fractal dimension) with a semiautomated computer-based program following a standardized protocol.

Results.: HIV-patients had more tortuous arterioles (0.77 × [10⁴] vs. 0.59 × [10⁴], P < 0.001) and venules (0.90 × [10⁴] vs. 0.74 × [10⁴], P < 0.001), compared with healthy normal subjects. Amongst the HIV-patients, increasing HIV viral loads were associated with decreased retinal arteriolar caliber (P trend = 0.009) and decreased arteriolar-venular ratio (P trend = 0.025).

Conclusions.: Our study showed that patients with HIV have significant variations in retinal vasculature. Retinal vascular imaging may offer further insight into the pathophysiology behind HIV-related vascular disease in future.

Introduction

Human immunodeficiency virus (HIV) is a significant global health problem. The 2010 Joint United Nations Programme on HIV/AIDS report estimated 34 million people are living with HIV, ¹ although global healthcare reforms and improvements in healthcare programs have resulted in decreased new incidences and HIV-related deaths. 

The retina allows in vivo noninvasive visualization of the microcirculation, and offers great promise in the monitoring of systemic microangiopathies. Previous studies have shown that early subclinical changes in the retinal microvasculature, not detectable on routine clinical examination, allow for the early detection of individuals at risk of developing microvascular diseases such as diabetes ^2,3 and hypertension. ^4,5 This has also led to the development of novel expertise in analyzing subclinical retinal microvascular geometric parameters (branching angles, bifurcation, fractal dimension, and tortuosity), which are more indicative of the overall “optimality state” of the retina. ⁶  

It is well established that HIV causes retinal microangiopathy. ^7–9 Previous literature has reported variations in retinal vascular caliber associated with AIDS-specific factors, which are markers for increased mortality risk. ¹⁰ In this study, we further examine the effects of HIV infection on the novel retinal vascular parameters mentioned above, which may provide additional insights into microvascular abnormalities in HIV. 

Methods

Study Population

This is a case control study conducted at the outpatient ophthalmology clinic at the Singapore Communicable Disease Centre. Patients who were HIV positive attending the clinic from June 2011 to June 2012 were included in the study. Patients with opportunistic ocular infections or media opacities that prevented the capture of good quality retinal images were excluded from the study. Two hundred and fifty-one normal controls matched for age, sex, race, comorbidities (hypertension, diabetes mellitus, ischemic heart disease, cerebrovascular accidents) were selected from studies under the SEED program, including the Singapore Chinese Eye Study, the Singapore Indian Eye Study, and the Singapore Malay Eye Study. Informed consent was obtained from all patients after explanation of the nature and possible consequences of the study. All study procedures were performed in accordance with the tenets of the Declaration of Helsinki as revised in 1989 and were approved by the Institutional Review Board of the National Healthcare Group. 

Clinical Examination

All patients underwent a clinical examination consisting of: best-corrected logMAR visual acuity, intraocular pressure measurement, anterior segment examination, and dilated fundus examination. We collected baseline demographic, medical history (including comorbidities of hypertension, diabetes mellitus, previous cerebrovascular accidents, and ischemic heart disease), and laboratory information. All patients were also screened for hypertension and diabetes by infectious disease physicians prior to enrollment into the study with blood pressure measurements and glycated sugar level measurements. There were six patients with hypertension, two patients with diabetes mellitus, and one patient with previous cerebrovascular accident. HIV-related information included time since HIV diagnosis, total white and lymphocyte count, CD4⁺ T-lymphocyte count, CD8+ T lymphocyte count, and HIV RNA blood level. Information on treatment including history and duration of highly active antiretroviral therapy (HAART) was also included. For purposes of this study, HAART was defined as the concurrent use of three or more antiretroviral drugs of which at least one of the drugs is a protease inhibitor. ¹¹  

Retinal Photography

The methods for obtaining digital fundal photographs and measuring retinal vascular caliber have been described earlier. ¹² Digital fundus photography was performed using a 45° retinal camera (Canon CR-DGi; Canon, Tokyo, Japan) with a digital camera back (10D SLR; Canon) after pharmacological dilation with tropicamide 1% eye drops. Two retinal images of each eye were obtained, one centered at the optic disc and another centered at the fovea. We used an optic disc-centered photograph of the right eye of each participant. If the photograph of the right eye was ungradable, the measurement was performed on the left eye. Ungradable photographs were secondary to media opacity or off-centered photographs. If first order vessels were not visible in the photograph, the patient was deemed to have ungradable photos due to media opacity. Off-centered photographs were defined in instances where the 2-disc diameter marking was more than one quadrant away from the retinal image. 

Quantitative Measurements of Retinal Microvasculature

We used a semiautomated computer-assisted program (Singapore I Vessel Assessment [SIVA], version 3.0; National University of Singapore, Singapore) to quantitatively measure a range of retinal vascular parameters (vascular caliber, vascular tortuosity and branching angles) from digital photographs according to a standardized protocol. Images of poor quality, including those due to media opacities (e.g., dense lens opacity), small size of the pupil, or images that were out of focus or that had poor contrast, were excluded. The measured area of retinal vascular branching, retinal vascular fractal dimension, and retinal vascular tortuosity was standardized and defined as the region from 0.5 to 2.0 disc diameters away from the disc margin. Three trained graders followed a standardized protocol and performed visual evaluations of the automated measurements with corrections made manually if necessary. ³ The graders were masked to the subjects' identity and other measured parameters in the Singapore Advanced Imaging Laboratory Research. One person graded each photograph and any ambiguous photographs were discussed among the three graders. Inter and intraobserver reliability measurements of the trained graders have been previously performed. ¹³  

Retinal Vascular Branching.

Retinal branching angle is defined as the first angle subtended between two daughter vessels at each vascular bifurcation. ¹⁴  

Retinal Vascular Fractal Dimension.

Fractal dimension was calculated from the skeletonized line tracing using a box-counting method; a “global” measure summarizing the whole branching pattern of the retinal vascular tree. ^14,15 Larger values indicated a more complex branching pattern. 

Retinal Vascular Tortuosity.

Retinal vascular tortuosity is defined as the integral of the curvature square along the path of the vessel, normalized by the total path length. ^3,16,17 A smaller tortuosity value indicates a straighter vessel. The estimates were summarized as retinal arteriolar tortuosity and retinal venular tortuosity, representing the average tortuosity of arterioles and venules of the eye, respectively. 

Retinal Vascular Caliber.

Retinal vascular caliber was measured following a standardized protocol, based on the revised Knudtson-Parr-Hubbard formula, as described in other publications. ¹⁸ A pair of indices, the central retinal arteriolar and venular equivalents (CRAE and CRVE), representing the average arteriolar and venular calibers for each eye, was then calculated. Arteriolar-venous ratio (AVR) was also calculated. 

Statistical Analysis

All statistical analysis was performed using statistical software (SPSS version 17.0; SPSS, Inc., Chicago, IL). Results between HIV patients were compared against age-, sex-, and race-matched normal controls. Measures of retinal vascular parameters were analyzed as continuous variables. Differences in continuous variables were analyzed using the independent sample t-test. Analysis of covariance was used to estimate mean retinal vessel geometric parameters by quartiles of viral load counts and CD4 counts in HIV patients. Test of trend was determined by treating categorical risk factors as continuous ordinal variables. 

Results

One hundred patients were recruited in this study but only eighty-five HIV patients had gradable fundal photographs. Twelve photographs were obscured by media opacity while three were off-centered photographs. Table 1 shows the demographic characteristics. Majority of patients were male (94.1%), consistent with the demographic trend in Singapore. ¹⁹ Median age of patients was 44 years (range; 26–64 years). The median duration of disease since initial diagnosis of HIV and HAART therapy was 7 months (range; 1–94 months). Eighty-three patients were on HAART therapy on enrollment in the study. The median CD4⁺ T lymphocytes count was 91 cells/μL (range; 15–952 cells/μL). 

Table 1

 

Study Demographics

Demographics	Patients	Controls	P Value
Age at baseline, mean, y	44.21	45.33	0.372
Sex, M/F (%)	80/5 (94.1/5.9)	242/9 (96.4/3.6)	0.372
AIDS-related patient characteristics, median (range)
Age at diagnosis  of HIV, y	42 (25–64)
Duration of HIV  since diagnosis,  mo	7 (1–94)
CD4 count,  cells/μL	91 (15–952)
Duration of  HAART, mo	7 (1–94)

Table 2 shows the quantitative retinal vascular measures (vessel caliber, branching angle, branching asymmetry ratio, fractal dimension, and tortuosity) of HIV patients and 251 age-, sex-, and race-matched controls (1:3 ratio). HIV patients had significantly more tortuous arterioles (0.77 [×10⁴] vs. 0.59 [×10⁴], P < 0.001) and venules (0.90 vs. 0.74, P < 0.001) compared with controls. There were no significant differences in CRAE, CRVE, arteriolar- venular ratio, vascular branching angle, and fractal dimension between HIV patients and controls. 

Table 2

 

Retinal Vascular Parameters

Retinal Vascular Parameters	Patients, n = 85		Controls, n = 251		P Value
	Mean	Standard Deviation	Mean	Standard Deviation
CRAE, μm	135.09	13.80	132.86	12.77	0.173
CRVE, μm	200.32	18.91	199.37	20.30	0.705
AVR	0.68	0.06	0.67	0.05	0.211
Arteriolar branching angle, deg	77.31	11.03	80.06	9.45	0.027
Venular branching angle, deg	79.27	9.38	79.16	10.42	0.931
Fractal dimension	1.45	0.05	1.45	0.04	0.648
Arteriolar tortuosity, ×104	0.77	0.13	0.59	0.17	<0.001
Venular tortuosity, ×104	0.90	0.13	0.74	0.21	<0.001

n  = 85 patients and 251 population-matched controls.

The relationships between retinal vascular parameters and HIV-related factors of HIV RNA viral loads and CD4 counts are shown in Table 3. Increasing serum levels of HIV RNA viral loads were significantly associated with decreased CRAE (P = 0.009) and decreased AVR (P = 0.025), after adjusting for venular caliber. There was no significant correlation of viral load with fractal dimension, branching angles or vascular tortuosity. There were no significant associations between change in CD4 counts or duration of HAART therapy and retinal vascular parameters. 

Table 3

 

Relationships Between Retinal Vascular Parameters and Laboratory Factors

	n	CRAE, μm	CRVE, μm	AVR	Fractal Dimension	Arteriolar Tortuosity, ×104	Arteriolar Branching Angle, deg	Venular Tortuosity, ×104	Venular Branching Angle, deg
Viral load counts log
First quartile	13	139.86, 2.56	195.97, 4.31	0.70, 0.01	1.4573, 0.0103	0.79, 0.04	79.86, 2.84	0.90, 0.03	81.46, 2.35
Second quartile	14	136.74, 2.48	197.47, 4.10	0.69, 0.02	1.4451, 0.0119	0.72, 0.02	76.46, 2.43	0.89, 0.04	76.11, 3.46
Third quartile	13	136.86, 2.61	204.70, 4.33	0.67, 0.01	1.4474, 0.0156	0.84, 0.05	79.14, 4.10	0.94, 0.03	78.99, 1.68
Fourth quartile	13	129.50, 2.56	206.15, 4.46	0.65, 0.02	1.4607, 0.0110	0.74, 0.02	78.32, 3.13	0.87, 0.02	81.16, 2.23
P trend		0.009	0.061	0.025	0.826	0.896	0.892	0.89	0.865
CD4 counts
First quartile	10	132.40, 1.92	203.14, 2.68	0.66, 0.01	1.4604, 0.01	0.76, 0.02	76.38, 2.19	0.91, 0.02	79.8, 1.71
Second quartile	18	137.43, 2.48	198.72, 3.46	0.69, 0.01	1.4492, 0.0115	0.80, 0.03	82.06, 1.93	0.88, 0.03	80.10, 2.37
Third quartile	15	133.88, 2.73	198.47, 3.80	0.68, 0.02	1.4380, 0.0108	0.73, 0.02	75.41, 3.21	0.85, 0.02	79.18, 2.25
Fourth quartile	12	137.69, 2.24	199.04, 3.14	0.69, 0.02	1.4516, 0.0101	0.79, 0.03	75.98, 2.18	0.93, 0.03	77.93, 2.09
P trend		0.202	0.351	0.152	0.39	0.742	0.433	0.941	0.454

n   = 85 patients.

Discussion

Our study aimed to examine the changes in retinal vascular parameters related to HIV infection and found that HIV patients have significant differences in vascular tortuosity compared to population-matched controls. Increasing levels of serum HIV RNA viral loads was also significantly associated with narrower arteriolar caliber and decreased AVR. 

Although previous studies have found increased vascular tortuosity to be linked to diabetes mellitus, ²⁰ anemia, ²¹ higher blood pressure, ^5,22 prematurity in preterm children, ²³ stroke, ²⁴ and dementia, ²⁵ our novel findings have not been previously reported in HIV patients. In our study, HIV patients were found to have increased arteriolar and venular tortuosity values compared with controls as depicted in the Figure. 

Figure

 

Retinal fundus photographs assessed quantitatively with ophthalmic software (National University of Singapore). Arterioles are in red and venules are in blue. The measured area of retinal vascular parameters (caliber, fractal dimension, tortuosity, and branching angle) was standardized as the region from 0.5 to 2.0 disc diameters away from the disc margin. (A) Retinal fundus photography of an HIV patient, demonstrating more tortuous retinal vessels in the patient with HIV. (B) Retinal fundus photograph of a control subject.

HIV-related endothelial dysfunction is well documented, but the mechanism is still unclear. Postulations include lipid deposition associated with HIV infection, ²⁶ viral protein-related endothelial activation, ²⁷ or effects of systemic inflammatory cytokine or chemokine dysregulation. HAART therapy has also been linked with increased atherosclerotic risk. ²⁸ We speculate that HIV-related endothelial dysfunction (regardless the mechanism) might induce a state of hypoxia resulting in subclinical retinal vascular parametric changes of increased tortuosity, complex branching patterns as above, and clinical features of retinal microangiopathy. Future laboratory studies involving retinal blood flow and oxygen levels may be useful to further determine this relationship. 

Tissue hypoxia results in a complex response mechanism mediated by secretagogues from vascular endothelial cells. The endothelial cells lining vessel walls play an important role in autoregulating blood flow by secreting mediators such as nitric oxide ²⁹ and endothelin. ³⁰ These chemicals stimulate angiogenesis and in turn increase tortuosity, which subsequently promotes improved tissue perfusion. ³¹  

A complex interaction of hemorheologic factors (abnormal erythrocyte aggregation, ³² leukocyte activation, ³³ abnormal vessel wall shear, ³⁴ abnormal rigidity of polymorphonuclear leucocyte ³⁵ ) previously reported in HIV patients could also lead to altered blood flow and ischemia. This could also result in subclinical retinal vascular parametric changes of increased tortuosity and more complex branching patterns as above, and clinical features of retinal microangiopathy. 

Advanced HIV disease is associated with higher baseline plasma viral load and a lower CD4⁺ T-lymphocyte count. ³⁶ Higher viral loads whilst on HAART was found to be associated with narrower arterioles and has been postulated to be secondary to inflammation as previously found in diabetes mellitus. ^2,36 Our study substantiates this finding and further noted decreasing AVR with increasing levels of HIV RNA viral loads. We did not manage to find a consistent significant difference between HIV patients and controls in terms of vessel caliber. We are unable to explain this discrepancy and future longitudinal studies with larger sample sizes will be required. 

We did not find any significant associations between CD4 counts and retinal vascular parameters. This was similar to findings from Pathai et al. ³⁷ who found no association. Gangaputra et al. ¹⁰ did, however, find an association between CD4 counts and decreased CRAE. Retinal microvasculature may be reflective of long-term changes while CD4 counts are a static and temporary measure of the patient's immune status and disease state. Thus, the two indices may not be correlates of each other. 

Although previous studies reported that retinal arterioles narrow with increasing duration of HAART, ³⁷ our study did not find any correlations between duration of HAART therapy and retina vascular parametric abnormalities. A few possible explanations could explain our findings. First, the median duration of HAART in this study was 7 months, compared with previous studies where retinal arteriolar narrowing was only found after HAART therapy for more than 6 years. ³⁷ Second, severe endothelial dysfunction has been documented previously in patients who received long-term protease-inhibitor based (especially indinavir) HAART, but not in those without a protease inhibitor. In comparison, the patients in our study received a combination of several drugs namely tenofovir, atazanavir, ritonavir, efavirenz, nevirapine, but not indinavir. ³⁸  

The strengths of this study include standardized assessment of retinal images, and quantitative measurement of retinal vasculatures with a computer-assisted program. Progress in retinal vascular imaging technologies in the last two decades has provided clinicians and researchers with a means to measure and quantify subtle variations and abnormalities in the retinal microvasculature. These new technologies have linked retinal microvascular signs with a greater risk of important clinical and subclinical cardiovascular/atherosclerotic outcomes. Comparisons with population-matched controls in a 1:3 ratio strengthened the analysis as well. Our study has a few limitations. The cross-sectional nature of our study prevents examination of causal relationships. A prospective study would be useful to elicit the relationships between retinal vascular parameters and mortality risks. Despite the standardized protocols used, the retinal vasculature grading includes measurement errors related to subjective grader input, variability in image quality and other unknown issues (e.g., pulse cycle) which may lead to misclassification or less precision of the measurement. Lipids might influence retinal vasculature, but we did not have serum lipid values for all patients. 

In conclusion, our study showed that patients with HIV have significant, unique variations in retinal vasculature. We demonstrate the presence of increased tortuosity in retinal vasculature of HIV patients. The question now lies in whether these vascular variations can predict increased risk of mortality in this group of patients as well as occurrence of ocular diseases like glaucoma or vein occlusions. Future clinical directions include prospective studies to determine which of these vessel caliber indices will be most useful clinically especially in predicting adverse events in patients with HIV and exploring the possibilities of retinal vascular imaging as a tool in HIV-related vascular risk stratification. 

Acknowledgments

Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2012. 

Supported by National Healthcare Group Small Innovative Group SIG No. 110116. The authors alone are responsible for the content and writing of the paper. 

Disclosure: P.B. Tan, None; O.K. Hee, None; C. Cheung, None; T.K. Yeo, None; R. Agrawal, None; J. Ng, None; T.H. Lim, None; T.Y. Wong, None; S.C. Teoh, None