- Email: [email protected]
Aqueous Flare and Choroidal Thickness in Patients with Chronic Hepatitis C Virus Infection
Aqueous Flare and Choroidal Thickness in Patients with Chronic Hepatitis C Virus Infection A Pilot Study Ernesto Strobbe, MD, Mauro Cellini, MD, Emilio C. Campos, MD Purpose: To investigate the status of the bloodeaqueous barrier and to evaluate the subfoveal choroidal thickness (SCT) in patients with asymptomatic untreated chronic hepatitis C virus (HCV) infection without any anterior or posterior ocular involvement and to search for possible correlations. Design: Observational case-control study. Participants and Controls: A total of 80 eyes of 20 HCV-positive patients (male-to-female ratio, 12:8; mean age, 46.97.23 years) and 20 healthy controls (male-to-female ratio, 10:10; mean age, 48.28.71 years) were examined. Methods: Participants underwent a complete ophthalmologic examination. Aqueous flare was quantified objectively by using the noninvasive laser flare cell meter FC-500 (Kowa Company Ltd, Tokyo, Japan), whereas SCT was evaluated by using enhanced depth imaging optical coherence tomography (Spectralis OCT; Heidelberg Engineering GmbH, Heidelberg, Germany). A Wilcoxon rank-sum test was performed to compare ocular findings between HCV patients and controls, and correlations were assessed by using the Spearman rank test. Main Outcome Measures: Retinal and choroidal thickness and anterior chamber inflammation of HCV patients and healthy controls. Results: Patients with HCV showed significantly higher aqueous flare values (8.372.25 photon counts/ms vs. 4.561.45 photon counts/ms; P<0.0001) and a significantly increased SCT (362.746.5 mm vs. 320.2532.82 mm; P<0.0001) than healthy controls. Moreover, subjects with liver fibrosis had higher flare values than those with no significant hepatic fibrosis (9.621.99 photon counts/ms vs. 6.972.19 photon counts/ms; P ¼ 0.0003) and thicker choroids (379.1544.75 mm vs. 346.343.27 mm; P ¼ 0.024). Statistical analysis revealed that there was a positive correlation between aqueous flare values and SCT in HCV patients (r ¼ 0.69; P<0.0001) and between flare and the degree of liver fibrosis (r ¼ 0.67; P ¼ 0.0001). Conclusions: This study showed that impairment of the bloodeaqueous barrier and thickened choroids are features of asymptomatic HCV patients, and that choroidal thickness increases as the degree of subclinical inflammation of the anterior chamber increases. Patients with significant liver fibrosis have the highest flare values and the thickest choroids. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2013;120:2258-2263 ª 2013 by the American Academy of Ophthalmology.
Hepatitis C virus (HCV) infection is very common, with an estimated 180 million patients affected worldwide.1 It is characterized by an asymptomatic nature of early infection and by a slow progression that may lead to severe hepatic complications, such as inflammation, necrosis, fibrosis, cirrhosis, or hepatocellular carcinoma. In particular, a diffuse infiltration of inflammatory cells and mediators into the liver parenchyma seems to play a critical role for the onset of fibrosis. The high prevalence of the chronic form of hepatitis C is the result of the rapid viral replication and the capacity to escape immune response2 and may result in a multitude of extrahepatic manifestations that can affect several organ systems with significant morbidity.3 Because little is known about the effect of HCV infection on the eye, it is of great importance to understand better how the virus may interact with ocular tissues. Indeed, in
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2013 by the American Academy of Ophthalmology Published by Elsevier Inc.
the literature, no pathognomonic ocular manifestation of HCV infection has been demonstrated. However, cotton wool spots, retinal hemorrhages, visual impairment, and more generalized ischemic retinopathy or ischemic optic neuropathy are features that appear more frequently in HCV patients undergoing interferon or combined therapy.4,5 However, HCV itself seems to be associated with idiopathic retinopathy6 and with various ocular external manifestations such as scleritis, keratitis,7 and dry eye8 through incompletely elucidated mechanisms in which inflammation seems to play a key role. In an attempt to establish better whether a relationship between HCV infection and ocular inflammation may exist and whether affected patients may show some abnormalities in the macula and choroid, we assessed the status of the bloodeocular barrier in chronic untreated HCV patients by ISSN 0161-6420/13/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.03.040
Strobbe et al
Ocular Subclinical Inflammation in HCV Patients
using the laser flare-cell meter and evaluated foveal central thickness and subfoveal choroidal thickness (SCT) by using spectral-domain optical coherence tomography.
Methods Subject Recruitment The current study was an observational, case-control, single-center study performed in accordance with the recommendations of the Declaration of Helsinki and was conducted between April 2012 and September 2012. Approval was obtained from the Institutional Review Board and Ethical Committee of University of Bologna. All patients were informed about the noninvasive nature of the examinations and consented to the procedures by signing a written consent. Twenty white subjects with HCV infection, followed up by the Gastroenterology Service for no more than 12 months, who were referred to the Ophthalmology Unit for a consultation to exclude any sign of ocular inflammation, retinopathy, or optic neuropathy before starting interferon or a combined therapy were selected from a cohort of 53 patients and were compared with 20 age-matched healthy controls who showed serologically negative results for hepatitis B and C infections and who were not taking any drug. Both Gastroenterology and Ophthalmology Services are part of the S. Orsola-Malpighi University Hospital, Bologna, Italy. Hepatitis C virus patients were required to meet the following inclusion criteria: age 60 years or younger, asymptomatic chronic HCV infection, good physical health, and no ocular symptoms or signs. Subjects were excluded if they had signs of decompensated liver disease, such as cirrhosis, ascites, hepatic encephalopathy, portal hypertension, and hepatorenal syndrome; inflammatory diseases; anterior or posterior segment surgery; laser treatment or trauma; previous episodes of ocular inflammation; a history of intraocular tumors; topical and systemic drug use; diabetes; psoriasis; pseudoexfoliation syndrome; and choroidal neovascularization.
Baseline Measurements Hepatitis C virus subjects underwent a medical interview and a general physical examination by the gastroenterologists who also had performed a transient elastography to assess hepatic fibrosis by measuring liver stiffness. Fibrosis was divided in nonsignificant (no fibrosis, low and mild fibrosis) and significant (with or without cirrhosis) forms, according to the validated liver biopsy METAVIR scoring system9; at present, indeed, a strong correlation between liver stiffness values and histologic stages of fibrosis has been demonstrated fully.10 All participants underwent a complete ophthalmic examination including visual acuity, applanation intraocular pressure assessment, biomicroscopy of the anterior and posterior segment, foveal central thickness, SCT, and aqueous flare measurement (Table 1). Subfoveal choroidal thickness, foveal central thickness, and aqueous flare were assessed 20 to 30 minutes after pupillary dilation with 1 drop of 1% tropicamide by an expert ophthalmologist who was blinded to the condition of patients. Each participant was evaluated at the same time of the day to eliminate possible confounders that could affect the results of the measurements, such as the diurnal variation of choroidal thickness, as demonstrated by Tan et al.11
Aqueous Flare Measurement Laser flare-cell meter is an automated objective and noninvasive technique that enables rapid and reproducible measurement of cells and protein content (flare) in the aqueous humor,12,13 which are signs of inflammation of the anterior segment of the eye, by providing information about the status of the bloodeaqueous barrier (BAB).
Table 1. Demographic and Ocular Characteristics of Hepatitis C Virus Patients and Healthy Controls
Male Female Age (yrs) IOP (mmHg) Mean spherical equivalent refractive error (D) Visual acuity (decimals) FCT (mm) SCT (mm) Flare (pc/ms)
Hepatitis C Virus Patients
Controls
12 8 46.97.23 15.72.51 0.351.24
10 10 48.28.71 15.52.28 0.20.99
1.00.0 235.7519.68 362.746.50 8.372.25
1.00.1 235.0517.40 320.2532.82 4.561.45
P Value
0.72 0.79 0.13 1.00 0.90 <0.0001* <0.0001*
D ¼ diopters; FCT ¼ foveal central thickness; IOP ¼ intraocular pressure; pc/ms ¼ photon counts per millisecond; SCT ¼ subfoveal choroidal thickness. *Statistically significant difference.
The device we used was the laser flare-cell meter FC-500 (Kowa Company, Ltd, Tokyo, Japan), which uses a diode laser that is projected into the anterior chamber to scan a measurement window of 0.30.5 mm over 0.5 second.12 The amount of backscattered light by protein particles in the aqueous humor, which is proportional to the concentration and size of proteins, is detected by a photomultiplier and processed by a computer. The average of signals coming from above and below the measurement window (background signals) is subtracted from the signal obtained from inside the scanned window to provide a laser flare photometry measurement. The accuracy and reproducibility of the method have been shown in several studies by different groups.12,14,15 The coefficient of variation is less than 10% and measurements are independent of the examiner using the instrument. Seven measurements for each eye were obtained and averaged, and those with artifacts were eliminated. The results were expressed as photon counts per millisecond (pc/ms).
Foveal Central Thickness and Subfoveal Choroidal Thickness Measurement Spectral-domain optical coherence tomography is a noninvasive, widespread technique that allows in vivo cross-sectional highresolution visualization of retinal structures and choroid with a fast scanning speed.16,17 However, to enhance the quality of the images and to provide many details of the architecture of the choroid, a new technique called enhanced depth imaging has been developed in the recent years.18,19 Briefly, the optical coherence tomography device was positioned close enough to the eye that the choroid moved closer to the 0 delay line and an inverted image of the posterior pole of the eye was obtained. As previously described by Spaide et al18 and Margolis and Spaide,19 we selected the horizontal scan running directly through the center of the fovea of both eyes of each patient by using spectral-domain optical coherence tomography (Heidelberg Engineering GmbH, Heidelberg, Germany) with the enhanced depth imaging technique. Foveal central thickness was measured automatically as the vertical distance, across the foveola, the tiny depression in the very center of the macula, from the inner to outer retinal demarcation lines, by using the intrinsic retinal segmentation algorithm, whereas SCT was measured manually and was defined as the vertical distance from the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidescleral interface,
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Ophthalmology Volume 120, Number 11, November 2013 exactly below the foveola (Fig 1). The values were expressed in micrometers (mm).
Statistical Analysis All data were expressed as the mean standard deviation. A statistical analysis was carried out to assess the differences in ocular findings between HCV patients and controls and between HCV patients with and without liver fibrosis by using the Wilcoxon rank-sum test. A Spearman correlation test was used to evaluate the relationship between aqueous flare values and SCT measurements and between aqueous flare values and the degree of hepatic fibrosis. P values less than 0.05 were regarded as being statistically significant.
and healthy subjects with regard to subfoveal choroidal thickness (362.746.50 mm vs. 320.2532.82 mm; P<0.0001; Table 1). Mean aqueous flare was 8.372.25 pc/ms (range, 4.4e12.2 pc/ms) in HCV patients and 4.561.45 pc/ms (range, 1.6e6.4 pc/ms) in eyes of healthy subjects, and the difference between the 2 groups was highly significant (P<0.0001; Table 1). In addition, in HCV patients, flare values showed a positive correlation to both SCT (r ¼ 0.69; P<0.0001; Fig 2) and the presence of significant liver fibrosis (r ¼ 0.67; P ¼ 0.0001). Indeed, with regard to subgroup analysis, subjects with increased liver involvement had higher flare values than those with no significant hepatic fibrosis (9.621.99 pc/ms vs. 6.972.19 pc/ms; P ¼ 0.0003) and thicker choroids (379.1544.75 mm vs. 346.343.27 mm; P ¼ 0.024; Table 2).
Discussion Results A total of 40 eyes from 20 HCV patients (male-to-female ratio, 12:8; mean age, 46.97.23 years; range, 40e59 years) and 40 control eyes of 20 healthy subjects (male-to-female ratio, 10:10; mean age, 48.28.71 years; range, 39e60 years) were examined in this study (Table 1). Clinically, no participants showed anterior chamber inflammatory reaction (cells and flare) or abnormalities in the lens, vitreous, optic disc, or retina at the slit-lamp biomicroscopic examination. Best-corrected visual acuity, intraocular pressure values, and retinal central thickness did not differ significantly between HCV patients and controls (Table 1). There was a significant difference between HCV
Hepatitis C virus infection is a fairly common and insidiously progressive liver disease, characterized by chronic inflammation. It may also result in a multitude of extrahepatic disease processes affecting, among others, the small vessels, kidneys, skin, thyroid, and eyes.3 To the best of our knowledge, no data have been published concerning intraocular inflammation or retinal and choroidal features in chronic HCV patients. In this pivotal study, we evaluated objective ocular findings in asymptomatic, untreated HCV patients and compared the results with those of age-matched controls.
Figure 1. Representative enhanced depth imaging optical coherence tomography scans of 2 eyes: (A) hepatitis C virus patient and (B) control subject. There is a marked choroidal thickness difference between the 2 patients (410 vs. 295 mm, respectively). Choroidal thickness was measured as the distance between the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidesclera junction. For illustration purposes, the resultant images were reinverted.
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Figure 2. Scatterplot showing the correlation between flare values (photon counts per millisecond) and subfoveal choroidal thickness (SCT; in micrometers) in patients with hepatitis C virus infection (Spearman’s correlation coefficient, r ¼ 0.69; P<0.0001).
We demonstrated that HCV patients have an 83.5% increase in aqueous flare values and a 13.2% increase in choroidal thickness compared with healthy subjects. An increase in anterior chamber flare measurements reflects a leakage of serum proteins, as well as inflammatory molecules and cells, into the anterior segment that mainly is the result of instability or disruption of the BAB, which, under normal conditions, acts as a competent barrier. This leads to a change in aqueous protein composition, size, or shape, as well as a change in protein concentration. Currently, aqueous flare and the status of the BAB may be recorded objectively by using the noninvasive laser flarecell meter.12 Indeed, several laser flare photometry studies have involved patients with anterior or posterior uveitis20 to investigate disease mechanisms better and to monitor the response to treatment in patients with cytomegalovirus retinitis,21 postsurgical inflammation,22 diabetic retinopathy,23 uveal melanoma,24 age-related macular degeneration,25 and retinal vein occlusions.26 Thus, by means of this useful technique, we showed that HCV infection leads to a breakdown of the BAB that causes a local anterior subclinical inflammation that is not apparent clinically by slit-lamp biomicroscopy. To our knowledge, the exact mechanism by which ocular inflammation occurs in chronic asymptomatic HCV patients is not clear. However, most extrahepatic abnormalities Table 2. Ocular Characteristics of Hepatitis C Virus Patients with and without Significant Liver Fibrosis
Male Female IOP (mmHg) FCT (mm) SCT (mm) Flare (pc/ms)
Hepatitis C Virus Patients with No Significant Fibrosis
Hepatitis C Virus Patients with Fibrosis
P Value
6 4 15.552.39 236.521.61 346.343.27 6.972.19
6 4 15.92.71 23518.08 379.1544.75 9.621.99
0.66 0.81 0.024* 0.0003*
FCT ¼ foveal central thickness; IOP ¼ intraocular pressure; pc/ms ¼ photon counts per millisecond; SCT ¼ subfoveal choroidal thickness. *Statistically significant difference.
generally are believed to be secondary to the host immune response to the virus.3 Indeed, HCV subjects show increased production of immune complexes, which circulate through the bloodstream and are deposited in blood vessels, resulting in complement activation and complement-dependent cytotoxicity of endothelial cells.27 Other mechanisms of ocular inflammation may include the molecular mimicry involving particular HCV antigens that could determine a host response toward epithelial or endothelial cells or endotheliitis itself, as has been demonstrated in the portal tracts.28 Indeed, antieendothelial cell antibodies directed against a variety of antigens on endothelial cells may induce activation of endothelial cells, resulting in an upregulated expression of endothelial adhesion molecules, secretion of chemokines and cytokines, or both29 and may induce vessel injuries and vascular damage. Furthermore, HCV may induce endothelial damage, as suggested by finding HCV antigens in normal vascular endothelial cells,30 and may trigger apoptosis of endothelial cells.31 Patients with HCV also showed thicker subfoveal choroids compared with controls, and by analyzing optical coherence tomography images, we observed an important dilatation and expansion of the entire choroidal vascular architecture in every study patient with a marked dilatation, especially at the level of the outermost Haller layer of choroidal vessels in 15 patients (75%; Fig 3). Subfoveally thickened choroid also was observed in patients with Vogt-Koyanagi-Harada disease,32 where it was related to a choroidal inflammatory reaction or infiltration, and in subjects with central serous chorioretinopathy,33,34 in which disturbances of the choroidal circulation and abnormal choroidal hyperpermeability were highlighted by using indocyanine green angiography. Patients with HCV enrolled in our study did not have any signs of severe liver dysfunction that could justify a significant alteration of the function of the hepatocytes, which are responsible for the daily biosynthesis of proteins and albumin that play a pivotal role in determining plasma colloid oncotic pressure.35 For this reason, we suppose that a reduced oncotic pressure in the vessels with a resulting increased fluid movement from the capillaries into the tissue, according to Starlings’ equation, is not the cause of the thickening of the choroids in our patients. In our opinion, the expansion and dilatation of the choroidal vascular architecture and the choroidal thickening in HCV patients may be caused by inflammation. Indeed, inflammation usually induces vasodilation and increases blood flow and vascular permeability with an increased movement of plasma into the tissues, leading to edema.36 Thus, the choroidal thickening we found in HCV patients may be explained by the effect of intraocular inflammation on the choroidal vasculature, even if a viral action on choroidal circulation or on vessel hyperpermeability cannot be excluded. To strengthen this hypothesis, we demonstrated a positive correlation between aqueous flare and SCT (r ¼ 0.69), suggesting that choroidal thickness increases in proportion to the breakdown of the BAB. However, we have no data regarding indocyanine green angiography in these patients. Furthermore, we showed that patients with increased hepatic fibrosis have higher flare values and thicker choroids
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Figure 3. Representative enhanced depth imaging optical coherence tomography scans of 4 eyes of hepatitis C virus patients. There is an important dilatation and expansion of the entire choroidal vascular architecture, with a marked dilatation especially at the level of the outermost Haller’s layer of large vessels. Choroidal thickness was measured as the distance between the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidescleral junction. For illustration purposes, the resultant images were reinverted.
than those with no significant fibrosis (a 38% and 9.5% increase, respectively). An explanation of the positive correlation between the degree of liver fibrosis and aqueous flare (r ¼ 0.67) in HCV infection may be that as hepatic involvement worsens, systemic inflammatory processes enhance and contribute to worsened BAB breakdown with an increase in the passage of constitutive elements of serum into the anterior segment and consequent higher intraocular inflammation. However, further investigations are needed to clarify whether this hypothesis may be confirmed. In conclusion, we demonstrated that chronic asymptomatic untreated HCV patients have an impairment of the BAB and a thickening of the choroid compared with healthy subjects, that choroidal thickness increases as the degree of subclinical inflammation of the anterior chamber increases, and that subjects with significant liver fibrosis have the highest aqueous flare values and the thickest choroids. This study has some limitations. First, given that this was a pilot study, the sample was chosen from a selected cohort of young asymptomatic and untreated chronic HCV patients. It is likely that results may be more heterogeneous by considering a general HCV population (treated and untreated, with or without ocular signs) or different phases of the disease (active, chronic, recurrent). Second, we had
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limited data on the HCV patients (such as time of diagnosis and duration of infection, general inflammatory status, liver function test results, cryoglobulinemia, and HCV viral load) because they were referred to our unit only for a consultation but, we expect that, in the future, larger samples could be analyzed to collect more data on the association between the eye and HCV infection and to understand better the relationship between systemic and ocular inflammation.
References 1. Ghany MG, Strader DB, Thomas DL, Seef LB. Diagnosis, management and treatment of hepatitis C: an update. Hepatology 2009;49:1335–74. 2. Chen SL, Morgan TR. The natural history of hepatitis C virus (HCV) infection. Int J Med Sci 2006;3:47–52. 3. Himoto T, Masaki T. Extrahepatic manifestations and autoantibodies in patients with hepatitis C virus infection. Clin Dev Immunol 2012;2012:871401. 4. Sene D, Touitou V, Bodaghi B, et al. Intraocular complications of IFN-alpha and ribavirin therapy in patients with chronic viral hepatitis C. World J Gastroenterol 2007;13:3137–40. 5. Kim ET, Kim LH, Lee JI, Chin HS. Retinopathy in hepatitis C patients due to combination therapy with pegylated interferon and ribavirin. Jpn J Ophthalmol 2009;53:598–602.
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6. Abe T, Nakajima A, Satoh N, et al. Clinical characteristics of hepatitis C virus-associated retinopathy. Jpn J Ophthalmol 1995;39:411–9. 7. Kedhar SR, Belair ML, Jun AS, et al. Scleritis and peripheral ulcerative keratitis with hepatitis C virus-related cryoglobulinemia [letter]. Arch Ophthalmol 2007;125: 852–3. 8. Gumus K, Yurci A, Mirza E, et al. Evaluation of ocular surface damage and dry eye status in chronic hepatitis C at different stages of hepatic fibrosis. Cornea 2009;28:997–1002. 9. Bedossa P, Poynard T. METAVIR Cooperative Study Group. An algorithm for grading of activity in chronic hepatitis C. Hepatology 1996;24:289–93. 10. Castera L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005;128:343–50. 11. Tan CS, Ouyang Y, Ruiz H, Sadda SR. Diurnal variation of choroidal thickness in normal, healthy subjects measured by spectral domain optical coherence tomography. Invest Ophthalmol Vis Sci 2012;53:261–6. 12. Sawa M, Tsurimaki Y, Tsuru T, Shimizu H. New quantitative method to determine protein concentration and cell number in aqueous in vivo. Jpn J Ophthalmol 1988;32:132–42. 13. Ladas JG, Wheeler NC, Morhun PJ, et al. Laser flare-cell photometry: methodology and clinical applications. Surv Ophthalmol 2005;50:27–47. 14. Shah SM, Spalton DJ, Taylor JC. Correlations between laser flare measurements and anterior chamber protein concentrations. Invest Ophthalmol Vis Sci 1992;33:2878–84. 15. Yoshitomi T, Wong AS, Daher E, Sears ML. Aqueous flare measurement with laser flare-cell meter. Jpn J Ophthalmol 1990;34:57–62. 16. Ruggeri M, Wehbe H, Jiao S, et al. In vivo three-dimensional high-resolution imaging of rodent retina with spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci 2007;48:1808–14. 17. Wojtkowski M, Srinivasan V, Fujimoto JG, et al. Three-dimensional retinal imaging with high-speed ultrahigh-resolution optical coherence tomography. Ophthalmology 2005;112: 1734–46. 18. Spaide RF, Koizumi H, Pozzoni MC. Enhanced depth imaging spectral-domain optical coherence tomography. Am J Ophthalmol 2008;146:496–500. 19. Margolis R, Spaide RF. A pilot study of enhanced depth imaging optical coherence tomography of the choroid in normal eyes. Am J Ophthalmol 2009;147:811–5. 20. Guex-Crosier Y, Pittet N, Herbort CP. Evaluation of laser flare-cell photometry in the appraisal and management of intraocular inflammation in uveitis. Ophthalmology 1994;101: 728–35.
21. Magone MT, Nussenblatt RB, Whitcup SM. Elevation of laser flare photometry in patients with cytomegalovirus retinitis and AIDS. Am J Ophthalmol 1997;124:190–8. 22. Schauersberger J, Kruger A, Müllner-Eidenböck A, et al. Long-term disorders of the blood-aqueous barrier after smallincision cataract surgery. Eye (Lond) 2000;14:61–3. 23. Küchle M, Schönherr U, Nguyen NX, et al. Quantitative measurement of aqueous flare and aqueous “cells” in eyes with diabetic retinopathy. Ger J Ophthalmol 1992;1:164–9. 24. Castella AP, Bercher L, Zografos L, et al. Study of the bloodaqueous barrier in choroidal melanoma. Br J Ophthalmol 1995;79:354–7. 25. Kubota T, Motomatsu K, Sakamoto M, et al. Aqueous flare in eyes with senile disciform macular degeneration: correlation with clinical stage and area of neovascular membrane. Graefes Arch Clin Exp Ophthalmol 1996;234:285–7. 26. Miyake K, Miyake T, Kayazawa F. Blood-aqueous barrier in eyes with retinal vein occlusion. Ophthalmology 1992;99: 906–10. 27. Ferri C, Mascia MT. Cryoglobulinemic vasculitis. Curr Opin Rheumatol 2006;18:54–63. 28. Yeh MM, Larson AM, Tung BY, et al. Endotheliitis in chronic viral hepatitis: a comparison with acute cellular rejection and nonalcoholic steatohepatitis. Am J Surg Pathol 2006;30:727–33. 29. Del Papa N, Guidali L, Sironi M, et al. Anti-endothelial cell IgG antibodies from patients with Wegener’s granulomatosis bind to human endothelial cells in vitro and induce adhesion molecule expression and cytokine secretion. Arthritis Rheum 1996;39:758–66. 30. Sansonno D, Cornacchiulo V, Lacobelli AR, et al. Localization of hepatitis C virus antigens in liver and skin tissues of chronic hepatitis C virus-infected patients with mixed cryoglobulinemia. Hepatology 1995;21:305–12. 31. Bordron A, Dueymes M, Levy Y, et al. The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis. J Clin Invest 1998;101:2029–35. 32. Maruko I, Iida T, Sugano Y, et al. Subfoveal choroidal thickness after treatment of Vogt-Koyanagi-Harada disease. Retina 2011;31:510–7. 33. Imamura Y, Fujiwara T, Margolis R, Spaide RF. Enhanced depth imaging optical coherence tomography of the choroid in central serous chorioretinopathy. Retina 2009;29:1469–73. 34. Maruko I, Iida T, Sugano Y, et al. Subfoveal choroidal thickness after treatment of central serous chorioretinopathy. Ophthalmology 2010;117:1792–9. 35. Fanali G, di Masi A, Trezza V, et al. Human serum albumin: from bench to bedside. Mol Aspects Med 2012;33:209–90. 36. Weissman G. Inflammation: historical perspective. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: Basic Principles and Clinical Correlates. 2nd ed. New York: Raven Press; 1992:51–3.
Footnotes and Financial Disclosures Originally received: January 5, 2013. Final revision: March 11, 2013. Accepted: March 27, 2013. Available online: June 4, 2013. Manuscript no. 2013-35. Ophthalmology Unit, Department of Experimental, Diagnostic, and Specialty Medicine, Alma Mater Studiorum, University of Bologna, Bologna, Italy.
Supported by the University of Bologna (ECC-MIUR ex-60%), Bologna, Italy; by the Fondazione Banca del Monte di Bologna e Ravenna; and by the Fondazione Cassa di Risparmio di Bologna, Bologna, Italy. Correspondence: Ernesto Strobbe, MD, Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. E-mail: [email protected].
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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Hepatitis C virus (HCV) infection is very common, with an estimated 180 million patients affected worldwide.1 It is characterized by an asymptomatic nature of early infection and by a slow progression that may lead to severe hepatic complications, such as inflammation, necrosis, fibrosis, cirrhosis, or hepatocellular carcinoma. In particular, a diffuse infiltration of inflammatory cells and mediators into the liver parenchyma seems to play a critical role for the onset of fibrosis. The high prevalence of the chronic form of hepatitis C is the result of the rapid viral replication and the capacity to escape immune response2 and may result in a multitude of extrahepatic manifestations that can affect several organ systems with significant morbidity.3 Because little is known about the effect of HCV infection on the eye, it is of great importance to understand better how the virus may interact with ocular tissues. Indeed, in
2258
2013 by the American Academy of Ophthalmology Published by Elsevier Inc.
the literature, no pathognomonic ocular manifestation of HCV infection has been demonstrated. However, cotton wool spots, retinal hemorrhages, visual impairment, and more generalized ischemic retinopathy or ischemic optic neuropathy are features that appear more frequently in HCV patients undergoing interferon or combined therapy.4,5 However, HCV itself seems to be associated with idiopathic retinopathy6 and with various ocular external manifestations such as scleritis, keratitis,7 and dry eye8 through incompletely elucidated mechanisms in which inflammation seems to play a key role. In an attempt to establish better whether a relationship between HCV infection and ocular inflammation may exist and whether affected patients may show some abnormalities in the macula and choroid, we assessed the status of the bloodeocular barrier in chronic untreated HCV patients by ISSN 0161-6420/13/$ - see front matter http://dx.doi.org/10.1016/j.ophtha.2013.03.040
Strobbe et al
Ocular Subclinical Inflammation in HCV Patients
using the laser flare-cell meter and evaluated foveal central thickness and subfoveal choroidal thickness (SCT) by using spectral-domain optical coherence tomography.
Methods Subject Recruitment The current study was an observational, case-control, single-center study performed in accordance with the recommendations of the Declaration of Helsinki and was conducted between April 2012 and September 2012. Approval was obtained from the Institutional Review Board and Ethical Committee of University of Bologna. All patients were informed about the noninvasive nature of the examinations and consented to the procedures by signing a written consent. Twenty white subjects with HCV infection, followed up by the Gastroenterology Service for no more than 12 months, who were referred to the Ophthalmology Unit for a consultation to exclude any sign of ocular inflammation, retinopathy, or optic neuropathy before starting interferon or a combined therapy were selected from a cohort of 53 patients and were compared with 20 age-matched healthy controls who showed serologically negative results for hepatitis B and C infections and who were not taking any drug. Both Gastroenterology and Ophthalmology Services are part of the S. Orsola-Malpighi University Hospital, Bologna, Italy. Hepatitis C virus patients were required to meet the following inclusion criteria: age 60 years or younger, asymptomatic chronic HCV infection, good physical health, and no ocular symptoms or signs. Subjects were excluded if they had signs of decompensated liver disease, such as cirrhosis, ascites, hepatic encephalopathy, portal hypertension, and hepatorenal syndrome; inflammatory diseases; anterior or posterior segment surgery; laser treatment or trauma; previous episodes of ocular inflammation; a history of intraocular tumors; topical and systemic drug use; diabetes; psoriasis; pseudoexfoliation syndrome; and choroidal neovascularization.
Baseline Measurements Hepatitis C virus subjects underwent a medical interview and a general physical examination by the gastroenterologists who also had performed a transient elastography to assess hepatic fibrosis by measuring liver stiffness. Fibrosis was divided in nonsignificant (no fibrosis, low and mild fibrosis) and significant (with or without cirrhosis) forms, according to the validated liver biopsy METAVIR scoring system9; at present, indeed, a strong correlation between liver stiffness values and histologic stages of fibrosis has been demonstrated fully.10 All participants underwent a complete ophthalmic examination including visual acuity, applanation intraocular pressure assessment, biomicroscopy of the anterior and posterior segment, foveal central thickness, SCT, and aqueous flare measurement (Table 1). Subfoveal choroidal thickness, foveal central thickness, and aqueous flare were assessed 20 to 30 minutes after pupillary dilation with 1 drop of 1% tropicamide by an expert ophthalmologist who was blinded to the condition of patients. Each participant was evaluated at the same time of the day to eliminate possible confounders that could affect the results of the measurements, such as the diurnal variation of choroidal thickness, as demonstrated by Tan et al.11
Aqueous Flare Measurement Laser flare-cell meter is an automated objective and noninvasive technique that enables rapid and reproducible measurement of cells and protein content (flare) in the aqueous humor,12,13 which are signs of inflammation of the anterior segment of the eye, by providing information about the status of the bloodeaqueous barrier (BAB).
Table 1. Demographic and Ocular Characteristics of Hepatitis C Virus Patients and Healthy Controls
Male Female Age (yrs) IOP (mmHg) Mean spherical equivalent refractive error (D) Visual acuity (decimals) FCT (mm) SCT (mm) Flare (pc/ms)
Hepatitis C Virus Patients
Controls
12 8 46.97.23 15.72.51 0.351.24
10 10 48.28.71 15.52.28 0.20.99
1.00.0 235.7519.68 362.746.50 8.372.25
1.00.1 235.0517.40 320.2532.82 4.561.45
P Value
0.72 0.79 0.13 1.00 0.90 <0.0001* <0.0001*
D ¼ diopters; FCT ¼ foveal central thickness; IOP ¼ intraocular pressure; pc/ms ¼ photon counts per millisecond; SCT ¼ subfoveal choroidal thickness. *Statistically significant difference.
The device we used was the laser flare-cell meter FC-500 (Kowa Company, Ltd, Tokyo, Japan), which uses a diode laser that is projected into the anterior chamber to scan a measurement window of 0.30.5 mm over 0.5 second.12 The amount of backscattered light by protein particles in the aqueous humor, which is proportional to the concentration and size of proteins, is detected by a photomultiplier and processed by a computer. The average of signals coming from above and below the measurement window (background signals) is subtracted from the signal obtained from inside the scanned window to provide a laser flare photometry measurement. The accuracy and reproducibility of the method have been shown in several studies by different groups.12,14,15 The coefficient of variation is less than 10% and measurements are independent of the examiner using the instrument. Seven measurements for each eye were obtained and averaged, and those with artifacts were eliminated. The results were expressed as photon counts per millisecond (pc/ms).
Foveal Central Thickness and Subfoveal Choroidal Thickness Measurement Spectral-domain optical coherence tomography is a noninvasive, widespread technique that allows in vivo cross-sectional highresolution visualization of retinal structures and choroid with a fast scanning speed.16,17 However, to enhance the quality of the images and to provide many details of the architecture of the choroid, a new technique called enhanced depth imaging has been developed in the recent years.18,19 Briefly, the optical coherence tomography device was positioned close enough to the eye that the choroid moved closer to the 0 delay line and an inverted image of the posterior pole of the eye was obtained. As previously described by Spaide et al18 and Margolis and Spaide,19 we selected the horizontal scan running directly through the center of the fovea of both eyes of each patient by using spectral-domain optical coherence tomography (Heidelberg Engineering GmbH, Heidelberg, Germany) with the enhanced depth imaging technique. Foveal central thickness was measured automatically as the vertical distance, across the foveola, the tiny depression in the very center of the macula, from the inner to outer retinal demarcation lines, by using the intrinsic retinal segmentation algorithm, whereas SCT was measured manually and was defined as the vertical distance from the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidescleral interface,
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Statistical Analysis All data were expressed as the mean standard deviation. A statistical analysis was carried out to assess the differences in ocular findings between HCV patients and controls and between HCV patients with and without liver fibrosis by using the Wilcoxon rank-sum test. A Spearman correlation test was used to evaluate the relationship between aqueous flare values and SCT measurements and between aqueous flare values and the degree of hepatic fibrosis. P values less than 0.05 were regarded as being statistically significant.
and healthy subjects with regard to subfoveal choroidal thickness (362.746.50 mm vs. 320.2532.82 mm; P<0.0001; Table 1). Mean aqueous flare was 8.372.25 pc/ms (range, 4.4e12.2 pc/ms) in HCV patients and 4.561.45 pc/ms (range, 1.6e6.4 pc/ms) in eyes of healthy subjects, and the difference between the 2 groups was highly significant (P<0.0001; Table 1). In addition, in HCV patients, flare values showed a positive correlation to both SCT (r ¼ 0.69; P<0.0001; Fig 2) and the presence of significant liver fibrosis (r ¼ 0.67; P ¼ 0.0001). Indeed, with regard to subgroup analysis, subjects with increased liver involvement had higher flare values than those with no significant hepatic fibrosis (9.621.99 pc/ms vs. 6.972.19 pc/ms; P ¼ 0.0003) and thicker choroids (379.1544.75 mm vs. 346.343.27 mm; P ¼ 0.024; Table 2).
Discussion Results A total of 40 eyes from 20 HCV patients (male-to-female ratio, 12:8; mean age, 46.97.23 years; range, 40e59 years) and 40 control eyes of 20 healthy subjects (male-to-female ratio, 10:10; mean age, 48.28.71 years; range, 39e60 years) were examined in this study (Table 1). Clinically, no participants showed anterior chamber inflammatory reaction (cells and flare) or abnormalities in the lens, vitreous, optic disc, or retina at the slit-lamp biomicroscopic examination. Best-corrected visual acuity, intraocular pressure values, and retinal central thickness did not differ significantly between HCV patients and controls (Table 1). There was a significant difference between HCV
Hepatitis C virus infection is a fairly common and insidiously progressive liver disease, characterized by chronic inflammation. It may also result in a multitude of extrahepatic disease processes affecting, among others, the small vessels, kidneys, skin, thyroid, and eyes.3 To the best of our knowledge, no data have been published concerning intraocular inflammation or retinal and choroidal features in chronic HCV patients. In this pivotal study, we evaluated objective ocular findings in asymptomatic, untreated HCV patients and compared the results with those of age-matched controls.
Figure 1. Representative enhanced depth imaging optical coherence tomography scans of 2 eyes: (A) hepatitis C virus patient and (B) control subject. There is a marked choroidal thickness difference between the 2 patients (410 vs. 295 mm, respectively). Choroidal thickness was measured as the distance between the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidesclera junction. For illustration purposes, the resultant images were reinverted.
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Figure 2. Scatterplot showing the correlation between flare values (photon counts per millisecond) and subfoveal choroidal thickness (SCT; in micrometers) in patients with hepatitis C virus infection (Spearman’s correlation coefficient, r ¼ 0.69; P<0.0001).
We demonstrated that HCV patients have an 83.5% increase in aqueous flare values and a 13.2% increase in choroidal thickness compared with healthy subjects. An increase in anterior chamber flare measurements reflects a leakage of serum proteins, as well as inflammatory molecules and cells, into the anterior segment that mainly is the result of instability or disruption of the BAB, which, under normal conditions, acts as a competent barrier. This leads to a change in aqueous protein composition, size, or shape, as well as a change in protein concentration. Currently, aqueous flare and the status of the BAB may be recorded objectively by using the noninvasive laser flarecell meter.12 Indeed, several laser flare photometry studies have involved patients with anterior or posterior uveitis20 to investigate disease mechanisms better and to monitor the response to treatment in patients with cytomegalovirus retinitis,21 postsurgical inflammation,22 diabetic retinopathy,23 uveal melanoma,24 age-related macular degeneration,25 and retinal vein occlusions.26 Thus, by means of this useful technique, we showed that HCV infection leads to a breakdown of the BAB that causes a local anterior subclinical inflammation that is not apparent clinically by slit-lamp biomicroscopy. To our knowledge, the exact mechanism by which ocular inflammation occurs in chronic asymptomatic HCV patients is not clear. However, most extrahepatic abnormalities Table 2. Ocular Characteristics of Hepatitis C Virus Patients with and without Significant Liver Fibrosis
Male Female IOP (mmHg) FCT (mm) SCT (mm) Flare (pc/ms)
Hepatitis C Virus Patients with No Significant Fibrosis
Hepatitis C Virus Patients with Fibrosis
P Value
6 4 15.552.39 236.521.61 346.343.27 6.972.19
6 4 15.92.71 23518.08 379.1544.75 9.621.99
0.66 0.81 0.024* 0.0003*
FCT ¼ foveal central thickness; IOP ¼ intraocular pressure; pc/ms ¼ photon counts per millisecond; SCT ¼ subfoveal choroidal thickness. *Statistically significant difference.
generally are believed to be secondary to the host immune response to the virus.3 Indeed, HCV subjects show increased production of immune complexes, which circulate through the bloodstream and are deposited in blood vessels, resulting in complement activation and complement-dependent cytotoxicity of endothelial cells.27 Other mechanisms of ocular inflammation may include the molecular mimicry involving particular HCV antigens that could determine a host response toward epithelial or endothelial cells or endotheliitis itself, as has been demonstrated in the portal tracts.28 Indeed, antieendothelial cell antibodies directed against a variety of antigens on endothelial cells may induce activation of endothelial cells, resulting in an upregulated expression of endothelial adhesion molecules, secretion of chemokines and cytokines, or both29 and may induce vessel injuries and vascular damage. Furthermore, HCV may induce endothelial damage, as suggested by finding HCV antigens in normal vascular endothelial cells,30 and may trigger apoptosis of endothelial cells.31 Patients with HCV also showed thicker subfoveal choroids compared with controls, and by analyzing optical coherence tomography images, we observed an important dilatation and expansion of the entire choroidal vascular architecture in every study patient with a marked dilatation, especially at the level of the outermost Haller layer of choroidal vessels in 15 patients (75%; Fig 3). Subfoveally thickened choroid also was observed in patients with Vogt-Koyanagi-Harada disease,32 where it was related to a choroidal inflammatory reaction or infiltration, and in subjects with central serous chorioretinopathy,33,34 in which disturbances of the choroidal circulation and abnormal choroidal hyperpermeability were highlighted by using indocyanine green angiography. Patients with HCV enrolled in our study did not have any signs of severe liver dysfunction that could justify a significant alteration of the function of the hepatocytes, which are responsible for the daily biosynthesis of proteins and albumin that play a pivotal role in determining plasma colloid oncotic pressure.35 For this reason, we suppose that a reduced oncotic pressure in the vessels with a resulting increased fluid movement from the capillaries into the tissue, according to Starlings’ equation, is not the cause of the thickening of the choroids in our patients. In our opinion, the expansion and dilatation of the choroidal vascular architecture and the choroidal thickening in HCV patients may be caused by inflammation. Indeed, inflammation usually induces vasodilation and increases blood flow and vascular permeability with an increased movement of plasma into the tissues, leading to edema.36 Thus, the choroidal thickening we found in HCV patients may be explained by the effect of intraocular inflammation on the choroidal vasculature, even if a viral action on choroidal circulation or on vessel hyperpermeability cannot be excluded. To strengthen this hypothesis, we demonstrated a positive correlation between aqueous flare and SCT (r ¼ 0.69), suggesting that choroidal thickness increases in proportion to the breakdown of the BAB. However, we have no data regarding indocyanine green angiography in these patients. Furthermore, we showed that patients with increased hepatic fibrosis have higher flare values and thicker choroids
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Figure 3. Representative enhanced depth imaging optical coherence tomography scans of 4 eyes of hepatitis C virus patients. There is an important dilatation and expansion of the entire choroidal vascular architecture, with a marked dilatation especially at the level of the outermost Haller’s layer of large vessels. Choroidal thickness was measured as the distance between the hyperreflective line of the retinal pigment epitheliumeBruch’s membrane complex to the innermost hyperreflective line of the choroidescleral junction. For illustration purposes, the resultant images were reinverted.
than those with no significant fibrosis (a 38% and 9.5% increase, respectively). An explanation of the positive correlation between the degree of liver fibrosis and aqueous flare (r ¼ 0.67) in HCV infection may be that as hepatic involvement worsens, systemic inflammatory processes enhance and contribute to worsened BAB breakdown with an increase in the passage of constitutive elements of serum into the anterior segment and consequent higher intraocular inflammation. However, further investigations are needed to clarify whether this hypothesis may be confirmed. In conclusion, we demonstrated that chronic asymptomatic untreated HCV patients have an impairment of the BAB and a thickening of the choroid compared with healthy subjects, that choroidal thickness increases as the degree of subclinical inflammation of the anterior chamber increases, and that subjects with significant liver fibrosis have the highest aqueous flare values and the thickest choroids. This study has some limitations. First, given that this was a pilot study, the sample was chosen from a selected cohort of young asymptomatic and untreated chronic HCV patients. It is likely that results may be more heterogeneous by considering a general HCV population (treated and untreated, with or without ocular signs) or different phases of the disease (active, chronic, recurrent). Second, we had
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limited data on the HCV patients (such as time of diagnosis and duration of infection, general inflammatory status, liver function test results, cryoglobulinemia, and HCV viral load) because they were referred to our unit only for a consultation but, we expect that, in the future, larger samples could be analyzed to collect more data on the association between the eye and HCV infection and to understand better the relationship between systemic and ocular inflammation.
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Footnotes and Financial Disclosures Originally received: January 5, 2013. Final revision: March 11, 2013. Accepted: March 27, 2013. Available online: June 4, 2013. Manuscript no. 2013-35. Ophthalmology Unit, Department of Experimental, Diagnostic, and Specialty Medicine, Alma Mater Studiorum, University of Bologna, Bologna, Italy.
Supported by the University of Bologna (ECC-MIUR ex-60%), Bologna, Italy; by the Fondazione Banca del Monte di Bologna e Ravenna; and by the Fondazione Cassa di Risparmio di Bologna, Bologna, Italy. Correspondence: Ernesto Strobbe, MD, Department of Experimental, Diagnostic, and Specialty Medicine, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. E-mail: [email protected].
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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