Retina
Retinal Vascular Parameter Variations in Patients With Human Immunodeficiency Virus Petrina B. Tan,1 Owen K. Hee,1 Carol Cheung,2–4 Tun Kuan Yeo,1 Rupesh Agrawal,1 James Ng,1 Tock Han Lim,1 Tien Yin Wong,2,3 and Stephen C. Teoh1 1
Ophthalmology, National Healthcare Group Eye Institute, Tan Tock Seng Hospital, Singapore Singapore Eye Research Institute, Singapore National Eye Centre, Singapore 3Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 Centre for Quantitative Medicine, Duke-NUS Graduate Medical School, Singapore 2
Correspondence: Stephen C. Teoh, Department of Ophthalmology, 11 Jalan Tan Tock Seng, Singapore 308433; [email protected]. Submitted: August 18, 2013 Accepted: October 22, 2013 Citation: Tan PB, Hee OK, Cheung C, et al. Retinal vascular parameter variations in patients with human immunodeficiency virus. Invest Ophthalmol Vis Sci. 2013;54:7962– 7967. DOI:10.1167/iovs.13-13081
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 (CD4þ 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 3 [104] vs. 0.59 3 [104], P < 0.001) and venules (0.90 3 [104] vs. 0.74 3 [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 ¼ 0.009) and decreased arteriolar-venular ratio (P trend ¼ 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. Keywords: HIV, retinal vasculature, tortuosity
uman 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 diabetes2,3 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
H
Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
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 7962
IOVS j December 2013 j Vol. 54 j No. 13 j 7963
Retinal Microvasculature in HIV TABLE 1. Study Demographics Demographics
Patients
Controls
P Value
Quantitative Measurements of Retinal Microvasculature
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, CD4þ T-lymphocyte count, CD8þ 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
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 boxcounting 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.
Retinal Photography
Statistical Analysis
The methods for obtaining digital fundal photographs and measuring retinal vascular caliber have been described earlier.12 Digital fundus photography was performed using a 458 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.
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 racematched 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.
Age at baseline, mean, y 44.21 45.33 Sex, M/F (%) 80/5 (94.1/5.9) 242/9 (96.4/3.6) AIDS-related patient characteristics, median (range) Age at diagnosis of HIV, y Duration of HIV since diagnosis, mo CD4 count, cells/lL Duration of HAART, mo
0.372 0.372
42 (25–64)
7 (1–94) 91 (15–952) 7 (1–94)
approved by the Institutional Review Board of the National Healthcare Group.
Clinical Examination
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
IOVS j December 2013 j Vol. 54 j No. 13 j 7964
Retinal Microvasculature in HIV TABLE 2. Retinal Vascular Parameters Patients, n ¼ 85
Controls, n ¼ 251
Retinal Vascular Parameters
Mean
Standard Deviation
Mean
Standard Deviation
P Value
CRAE, lm CRVE, lm AVR Arteriolar branching angle, deg Venular branching angle, deg Fractal dimension Arteriolar tortuosity, 3104 Venular tortuosity, 3104
135.09 200.32 0.68 77.31 79.27 1.45 0.77 0.90
13.80 18.91 0.06 11.03 9.38 0.05 0.13 0.13
132.86 199.37 0.67 80.06 79.16 1.45 0.59 0.74
12.77 20.30 0.05 9.45 10.42 0.04 0.17 0.21
0.173 0.705 0.211 0.027 0.931 0.648
Retinal Vascular Parameter Variations in Patients With Human Immunodeficiency Virus Petrina B. Tan,1 Owen K. Hee,1 Carol Cheung,2–4 Tun Kuan Yeo,1 Rupesh Agrawal,1 James Ng,1 Tock Han Lim,1 Tien Yin Wong,2,3 and Stephen C. Teoh1 1
Ophthalmology, National Healthcare Group Eye Institute, Tan Tock Seng Hospital, Singapore Singapore Eye Research Institute, Singapore National Eye Centre, Singapore 3Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 Centre for Quantitative Medicine, Duke-NUS Graduate Medical School, Singapore 2
Correspondence: Stephen C. Teoh, Department of Ophthalmology, 11 Jalan Tan Tock Seng, Singapore 308433; [email protected]. Submitted: August 18, 2013 Accepted: October 22, 2013 Citation: Tan PB, Hee OK, Cheung C, et al. Retinal vascular parameter variations in patients with human immunodeficiency virus. Invest Ophthalmol Vis Sci. 2013;54:7962– 7967. DOI:10.1167/iovs.13-13081
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 (CD4þ 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 3 [104] vs. 0.59 3 [104], P < 0.001) and venules (0.90 3 [104] vs. 0.74 3 [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 ¼ 0.009) and decreased arteriolar-venular ratio (P trend ¼ 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. Keywords: HIV, retinal vasculature, tortuosity
uman 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 diabetes2,3 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
H
Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
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 7962
IOVS j December 2013 j Vol. 54 j No. 13 j 7963
Retinal Microvasculature in HIV TABLE 1. Study Demographics Demographics
Patients
Controls
P Value
Quantitative Measurements of Retinal Microvasculature
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, CD4þ T-lymphocyte count, CD8þ 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
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 boxcounting 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.
Retinal Photography
Statistical Analysis
The methods for obtaining digital fundal photographs and measuring retinal vascular caliber have been described earlier.12 Digital fundus photography was performed using a 458 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.
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 racematched 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.
Age at baseline, mean, y 44.21 45.33 Sex, M/F (%) 80/5 (94.1/5.9) 242/9 (96.4/3.6) AIDS-related patient characteristics, median (range) Age at diagnosis of HIV, y Duration of HIV since diagnosis, mo CD4 count, cells/lL Duration of HAART, mo
0.372 0.372
42 (25–64)
7 (1–94) 91 (15–952) 7 (1–94)
approved by the Institutional Review Board of the National Healthcare Group.
Clinical Examination
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
IOVS j December 2013 j Vol. 54 j No. 13 j 7964
Retinal Microvasculature in HIV TABLE 2. Retinal Vascular Parameters Patients, n ¼ 85
Controls, n ¼ 251
Retinal Vascular Parameters
Mean
Standard Deviation
Mean
Standard Deviation
P Value
CRAE, lm CRVE, lm AVR Arteriolar branching angle, deg Venular branching angle, deg Fractal dimension Arteriolar tortuosity, 3104 Venular tortuosity, 3104
135.09 200.32 0.68 77.31 79.27 1.45 0.77 0.90
13.80 18.91 0.06 11.03 9.38 0.05 0.13 0.13
132.86 199.37 0.67 80.06 79.16 1.45 0.59 0.74
12.77 20.30 0.05 9.45 10.42 0.04 0.17 0.21
0.173 0.705 0.211 0.027 0.931 0.648
