Role of Avastin in management of central serous chorioretinopathy
Article Outline
Abstract
Purpose
To evaluate the short-term safety and efficacy of intravitreal bevacizumab for the treatment of intraretinal or subretinal fluid accumulation secondary to central serous chorioretinopathy (CSC).
Design
Prospective interventional series non-comparative study.
Setting
Department of Ophthalmology, Al-Minya University Faculty of Medicine, Egypt.
Methods
The study included 20 eyes of 20 patients with central serous chorioretinopathy (CSC), Out of them 10 eyes with acute CSC (group I), 6 eyes with chronic CSC (defined as symptoms present for longer than 6
months) and four eyes with recurrent (defined as more than one episode of the disease) chronic and recurrent cases are considered in one group (group II), all patients were injected with intravitreal Avastin (IVA) 1.25mg (0.05mL) of commercially available bevacizumab [Avastin; Genentech, Inc., San Francisco, CA] as a primary treatment. At baseline and follow up visits patients had best corrected visual acuity (BCVA), IOP assessment, dilated fundus examination, fundus photography, fluorescein angiography (FA) and optical coherence tomography (OCT) imaging is used for measurement of central retinal thickness (CRT). Main outcome measures were the resolution of neurosensory detachment, improvement in visual symptoms and visual acuity, and resolution of leakage in FA. Secondary outcome and measures were the need for re-injection and the adverse effects. The mean number of injections was 2 (range 1–3 injections) 6–8weeks intervals and follow up for 6months (range 5–7months). All finding at baseline and each follow up visit were reported and compared.
Results
The mean age of all patients was 40.3years±6.5 (range 25–50years), 15 males and five females patients. In acute CSC group, the mean baseline BCVA was 20/60 (log MAR 0.48) and improved to 20/30 (log MAR 0.18) with statistically significance difference change (P<0.003) and in (chronic and recurrent group), the mean baseline VA was 20/80 (log MAR 0.60) and improved to 20/40 (log MAR 0.30) with statistically significance difference change (P<0.002). The mean baseline CRT for all patients was 486±86μm (range, 386–580), decreased to 316±56μm (range, 276–368) after 1months with statistically significance difference change (P<0.02) and to 272±52μm (range 220–338) at last follow up with statistically significance difference change from the baseline (P<0.001).
Conclusions
Intravitreal Avastin (IVA) injection was associated with visual improvement and reduced neurosensory detachment without adverse events in patients with CSC. Although these results are promising, further randomized controlled studies would be helpful to understand this therapy for patients with CSC.
Keywords: Central serous chorioretinopathy, Avastin (bevacizumab), FA, OCT, Intravitreal injection
1. Introduction
Central serous chorioretinopathy (CSC) is a disease of young and middle-aged adults as a localized detachment of the neurosensory retina. It frequently manifests symptomatically in one eye, while up to 18% of cases may be bilateral. Research indicates that the disease process in CSC is more diffuse and shows bilateral retinochoroidal dysfunction, even when the disease is manifesting clinically only in one eye. Though the cause of CSC remains unknown, it is believed that abnormalities in choroidal circulation make overlying retinal pigment epithelium dysfunctional, resulting in the development of a serous retinal detachment. Photodynamic therapy, laser photocoagulation and pharmacological agents (Acetazolamide, propanolol, Mifepristone and Ketoconazole) have been used to treat CSC. However, these treatment options serve only to shorten the duration of symptoms and have no effect on the recurrence rate or the final visual acuity (Schaal et al., 2009). In cases with chronic diffuse or persistent focal leakage, retinal pigment epithelium (RPE) may decompensate leading to gradual visual loss with a less favorable visual prognosis (Jalk et al., 1984, Loo et al., 2002).
Type A personalities, systemic hypertension, and obstructive sleep apnea may be associated with CSC. The pathogenesis here is thought to be elevated circulating cortisol and epinephrine, which affect the autoregulation of the choroidal circulation. Furthermore, (Tewari et al., 2006), demonstrated that patients with CSC showed impaired autonomic response with significantly decreased parasympathetic activity and significantly increased sympathetic activity. Corticosteroids have a direct influence on the expression of adrenergic receptor genes and, thus, contribute to the overall effect of catecholamines on the pathogenesis of CSC. Consequently, multiple studies have conclusively implicated the effect of corticosteroids in the development of CSC. Carvalho-Recchia et al. (2002) showed in a series that 52% of patients with CSC had used exogenous steroids within 1month of presentation as compared with 18% of control subjects.
Cotticelli et al. (2006) showed an association between Helicobacter pylori infection and CSC. The prevalence of H. pylori infection was 78% in patients with CSC compared with a prevalence of 43.5% in the control group. The authors proposed that H. pylori infection may represent a risk factor in CSC, though no further studies have substantiated this claim. CSC is not a benign disease, as RPE atrophy and associated visual loss in chronic or persistent cases is slow but continuous process (Jalk et al., 1984).
Cardillo Piccolino et al. (1995), reported by indocyanine green (ICG) angiography, primary choroidal involvement in CSC, consisting of areas of choroidal vascular hyperpermeability and hence leakage in the RPE and RPE decompensation. On the basis of this knowledge, it seems reasonable to target the choroidal vascular changes with new strategies to treat CSC.
This study was performed to evaluate the short-term safety and efficacy of IVA for the treatment of intraretinal or subretinal fluid accumulation secondary to different forms of CSC.
2. Patients and methods
This prospective interventional series non-comparative study included 20 eyes of 20 patients with CSC, 10 eyes (50%) had acute CSC and six eyes (30%) had chronic CSC (defined as symptoms present for longer than 6months) and four eyes (20%) with recurrent (defined as more than one episode of the disease). Inclusion criteria including; documented subfoveal fluid by OCT, active leak associated with the subfoveal fluid, the ability and willingness to provide written informed consent, no signs of choroidal neovascularization. Exclusion criteria including; prior treatment with laser photocoagulation, TTT (transpupillary thermotherapy) or PDT (photodynamic therapy), have a history of thromboembolic events including stroke, transient ischemic attacks, and myocardial infarction, IVT (intravitreal triamcinolone) or bevacizumab in the previous 2months, uncontrolled glaucoma (IOP >24mmHg) on greater than three medications, visual acuity of worse than 20/400 in the fellow eye, unable to follow or comply with all study related procedures, have uncontrolled hypertension, previously vitrectomized eyes or had allergic reactions to fluorescein dye. All eyes were injected with intravitreal Avastin (IVA) 1.25mg (0.05mL) of commercially available bevacizumab [Avastin; Genentech, Inc., San Francisco, CA] at a concentration of 25mg/mL.
At baseline and follow up visits patients had measured best corrected visual acuity (BCVA), IOP assessment, dilated fundus examination, slit lamb biomicroscopy with Volk 90 and 78 diopters lenses, fundus photography, fluorescein angiography (FA) and optical coherence tomography (OCT) imaging is used for measurement of central retinal thickness (CRT). Main outcome measures were the resolution of neurosensory detachment, improvement in visual symptoms and visual acuity, and resolution of leakage in FA. Secondary outcome measures were the need for re-injection and the adverse effects. Optical coherence tomography (OCT) was performed for all of patients. Stratus OCT3, (Carl Zeiss Meditec, Dublin, CA). Readings for 1mm central retinal thickness (CRT) were obtained from the mean retinal thickness in the central subfield using six linear scans 6mm long centered on fixation and processed as a retinal map.
In all patients, the intravitreal injection of off label bevacizumab was performed in a standard protocol in the operating theater under operating ophthalmoscope and complete aseptic condition after obtaining informed consent. Topical 0.4% Benoxinate hydrochloride was applied to the ocular surface followed by scrubbing of the eye lids and lashes with 10% of Povidone iodine and conjunctiva installed with 5% Povidone iodine three times several minutes apart. A sterile eyelid speculum was used for all injections, sub-conjunctival 1cc Lidocaine 2% was done at the site of bevacizumab injection in the inferotemporal quadrant. Bevacizumab was injected through the pars plana 3.5–4.0mm posterior to the surgical limbus using a 30-gauge needle at a dose of 1.25mg in 0.05mL. Post injection, a sterile cotton swab is placed at the site of injection to prevent reflux of vitreous or drug, light perception was assessed and indirect ophthalmoscope to see optic nerve head perfusion.
After the injection, the patients were instructed to apply topical antibiotics to the injected eye four times a day for 5days. Postoperative follow up included repeated clinical examinations and OCT to all of the patients. Patients were assessed for adverse events including elevated intraocular pressure, cataract progression, retinal detachment, post-injection inflammation, and endophthalmitis. Follow up evaluations were scheduled to next day, 1week then monthly till the end of follow up. Repeated FA and OCT were done at 1month, 3months and at the end of follow up, about 6months. A repeated injection of bevacizumab was performed for persistent or recurrent CSC seen by fluorescein angiography and documented by OCT imaging.
The main outcome included resolution of leakage in FA, changes in CRT using OCT and changes in BCVA. Secondary outcome included any systemic or ocular adverse effects of IVA injection and the need for repeated injections. Snellen’s BCVA were converted to logarithmic minimum angle of resolution (log MAR) for statistical analysis. Statistical analysis was assessed using (SPSS, version 13, SPSS Inc., Chicago, Illinois, USA). All variables were expressed as mean±standard deviation (SD), the paired Student t-test was used and P value ⩽0.05 was considered statistically significant.
3. Results
In this prospective study, 20 eyes of 20 patients received intravetrial Avastin (IVA) injection. Out of them 10 eyes (50%) with acute CSC (group I) and six eyes (30%) with chronic CSC (defined as symptoms present for longer than 6months) and four eyes (20%) with recurrent (defined as more than one episode of the disease), chronic and recurrent eyes with CSC considered in one group (group II). The mean age of all patients was 40.3years ±6.5 (range 25–50years), 15 males and five females patients. Fourteen patients (70%) out of all patients demonstrated positive (HP) H. pylori infection. No one had history of recent corticosteroid therapy. The mean follow up period was 6months (range, 5–7months).
In acute CSC group, the mean baseline BCVA was 20/60 (log MAR 0.48) and improved to 20/30 (log MAR 0.18) at the end of follow up with statistically significance difference change (P<0.003) and in (chronic and recurrent group), the mean baseline VA was 20/80 (log MAR 0.60) and improved to 20/40 (log MAR 0.30) at last follow up with statistically significance difference change (P<0.003). The overall mean of BCVA of all cases at baseline was 20/70 (log MAR 0.54) and improved to 20/35 (log MAR 0.24) at last follow up with statistically significance difference change (P<0.002) as shown in Table 1. All of eyes had visual improvement, 14 eyes (70%) had gained two or more lines improvement in BCVA at the end of follow up. None of eyes had decreased vision than the baseline BCVA.
Table 1. Mean CRT and mean BCVA at baseline and follow up.
| Timing | Mean (SD) {range} CMT (μm) | Dif. CMT (μm) | P value | Mean BCVA | Log MAR (SD) | P value |
|---|---|---|---|---|---|---|
| Baseline | 486 (±86) {386–580} | 20/70 | 0.54 (±0.25) | |||
| 1month | 316 (±56) {276–368} | 170 | <0.02 | 20/50 | 0.35 (±0.17) | <0.01 |
| 3months | 325 (±76) {288–380} | 161 | <0.02 | 20/50 | 0.35 (±0.17) | <0.01 |
| 6months | 272 (±52) {220–338} | 214 | <0.001 | 20/35 | 0.24 (±0.18) | <0.003 |
There was decrease in the mean of CRT all over the time which was correlated with the improvement in both of BCVA and FA leakage. The mean baseline CRT for all patients was 486±86μm (range, 386–580), decreased to 316±56μm (range, 276–368) after 1months with statistically significance difference change (P<0.02) and difference of 170μm from baseline CRT and to 325±76μm (range, 288–380) after 3months with a difference of 161μm and to 272±52μm (range 220–338) at last follow up with statistically significance difference change from the baseline (P<0.001) in which the difference was 214μm as shown in Table 1.
There was complete resolution of fluorescein leakage after one injection in eight eyes (40%) of all patients within 2weeks, out of them five eyes in group I and three eyes in group II. Twelve eyes (60%) had not resolved completely by one injection and needs re-injection or recurrence and re-injection was done. However, the leakage in recurrent eyes was with less severity than that of the baseline. Six eyes in group II and two eyes in group I need three IVA injections as demonstrated in Table 2.
Table 2. Mean, number of injections for each eye.
| Number of injections | 10 Eyes of group (I) CSC | 10 Eyes of group (II) CSC | Total number of eyes (%) |
|---|---|---|---|
| One injection | 5 | 3 | 8 (40%) |
| Two injections | 3 | 1 | 4 (20%) |
| Three injections | 2 | 6 | 8 (40%) |
| Total number of injection, (mean) | 17, (1.7) | 23, (2.3) | 40, (2) |
Table 2 reported the mean number of IVA injection for all patients, it was 2 (range, 1–3), 6–8week interval for re-injection and the total number of injections are 40 injections for 20 eyes. It was a little bit higher in group II in which it was (2.3), 23 injections for 10 eyes and a little bit less in group I in which it was (1.7), 17 injections for 10 eyes. There were 14 patients (70%) out of all patients’ demonstrated positive (HP) H. pylori infection by serum antibody titer and sometimes by urea breath test when available. There were neither systemic nor intraocular adverse effects seen in our study except some sort of sub-conjunctival hemorrhage at the site of injection resolved spontaneously with time in three patients.
4. Discussion
Central serous chorioretinopathy (CSC) is a disease in which a serous detachment of the neurosensory retina occurs over an area of leakage from the choriocapillaris through the retinal pigment epithelium (RPE). CSC may be divided into two distinct clinical presentations. Classically, acute CSC is caused by one or more discrete isolated leaks at the level of the RPE as seen on fluorescein angiography (FA). However, it is now recognized that CSC may present with diffuse retinal pigment epithelial dysfunction (e.g. diffuse retinal pigment epitheliopathy, chronic CSC, decompensated RPE) characterized by neurosensory retinal detachment overlying areas of RPE atrophy and pigment mottling. During FA, broad areas of granular hyperfluorescence that contain one or many decompensatory leaks are seen as in Fig. 3B. Previous hypotheses for the pathophysiology have included abnormal ion transport across the RPE and focal choroidal vasculopathy. The advent of indocyanine green (ICG) angiography has highlighted the importance of the choroidal circulation to the pathogenesis of CSC. ICG angiography has demonstrated both multifocal choroidal hyperpermeability and hypofluorescent areas suggestive of focal choroidal vascular compromise. Some investigators believe that initial choroidal vascular compromise subsequently leads to secondary dysfunction of the overlying RPE (Schaal et al., 2009).
Chappelow and Marmor (2000) reported in their study using multifocal electroretinography that CSC patients had bilateral diffuse retinal dysfunction even when CSC was active only in one eye and diminished response amplitudes even after complete absorption of subretinal fluid. These studies support the belief of diffuse systemic effect on the choroidal vasculature. Wang et al. (2002) reported that foveal atrophy in CSC is associated with reduction of visual acuity despite resolution of serous detachment and hence reattachment within 4months of onset is considered a relevant therapeutic target because prolonged detachment is associated with photoreceptor atrophy. Stewart (2006) reported that the fact that laser damages the RPE with a known risk of developing choroidal neovascularization (CNV) has left clinicians seeking new treatments that are both more effective and less toxic. Without prior ICG dye, investigators have also used the 810nm laser as transpupillary thermotherapy (TTT) with moderate anecdotal success. However, Penha et al. (2007) described severe retinal thermal injury in a 31-year-old man following this treatment modality and recommended caution for this adverse event following ICG mediated TTT treatment.
Cardillo Piccolino et al. (2003), concluded that, PDT guided by ICG angiography and performed according to the protocol of the TAP Study seems to resolve macular detachment, serous exudation and preserve visual acuity in patients with chronic CSC. PDT could reduce choriocapillaris blood flow and choroidal exudation in the areas of subretinal fluid production. However, undesired effects are pigmentary changes in the treatment zone and persistent hypoperfusion of the choriocapillaris. Chan et al. (2003), reported development of CNV within the photodynamic zone due to localized ischemia in choriocapillaries secondary to PDT in chronic CSC patients with unhealthy RPE.
In our study IVA was used for treatment of different forms of CSC patients as shown in Figure 1, Figure 2, Figure 3, Figure 4 and had good results inspit of there is no direct evidence for vascular endothelial growth factor (VEGF) in inducing CSC. Shams and Ianchulev (2006), reported that VEGF is a critical regulator of ocular angiogenesis and vascular permeability. When increased choroidal hyperpermeability claimed to be one of the main underlying mechanisms of CSC, it seemed logic to treat such cases with anti-VEGF such as (Avastin, Genentech, Inc., San Francisco, CA), a full-length recombinant monoclonal antibody against human vascular endothelial growth factor which was approved by the food and drug administration for treatment of colorectal cancer and used to treat different ocular diseases on off label basis with informed consent of patients.
Figure 1.
(A) Colored fundus photography of Rt. acute CSC at baseline. (B) OCT of the same patient with sensory retinal detachment and subretinal fluid and increased CRT
=480μm at baseline. (C) OCT of the same patient with incomplete or partial resolution of subretinal fluid and decreased CRT=280μm after 1month of IVA injection. (D) OCT of the same patient with complete resolution of subretinal fluid and decreased CRT=240μm after 1month of the second IVA injection at the end of follow up.
Figure 2.
Colored fundus photography of Rt., eye with recurrent CSC at baseline, subretinal fluid was turbid.
Figure 3.
(A) Colored fundus photography of Lt., eye with chronic CSC showing RPE atrophy at baseline. (B) FA, of the same case showing granular hyperfluorescence with many decompensatory leaks and two macular leaking spots in venous phase. (C) FA of the same case with fading in late stage.
Figure 4.
(A) FA, of acute case of CSC with active central leaking hyperfluorescent spot. (B) FA, of the same case with enlargement of the leaking spot. (C) FA, of the same case with more leaking, enlargement and smoke stack formation. (D) FA, of the same case with resolution of the leaking spot after 2
weeks of IVA injection.
In the current study, 14 patients (70%) out of all patients demonstrated positive (HP) H. pylori infection and this in agreement with the results of (Cotticelli et al., 2006), in which they reported an association between H. pylori infection and CSC, the prevalence of H. pylori infection was 78% in patients with CSC compared with a prevalence of 43.5% in the control group. Our result as well as that of the previous mentioned study in this point draws our attention to thoroughly investigate all patients of CSC for (HP) infection and to treat positive cases with proper medical treatment. As shown in Fig. 6, the overall mean of BCVA in our study improved from 20/70 at baseline to 20/50 after one month and stayed stationary for the third month after IVA injection (1.25mg) then improved more to 20/35 after 6months due to complete resolution of some cases in which it was previously had partial resolution as in Fig. 1C or recurrence and improved by re-injection as only 40% of patients had complete resolution after one IVA injection. Resolution and improvement of vision depend on the viscosity of subretinal fluid, duration of the disease and amount of photoreceptor damage. However all of eyes had visual improvement and 70% of patients had gained two or more lines improvement than the baseline. There was decrease in the mean of CRT all over the time which was correlated with the improvement in both of BCVA and FA leakage. The mean baseline CRT for all patients was 486±86μm (range, 386–580), decreased to 272±52μm (range 220–338) at last follow up with statistically significance difference change from the baseline (P<0.001) in which the difference was 214μm as demonstrated in Fig. 5.
Figure 5.
Graph for CRT over follow up time.
Figure 6.
Graph for BCVA over follow up time.
Our result in agreement with (Torres-Soriano et al., 2008), they reported in a study of six eyes (five chronic and one recurrent CSC) that visual acuity improved in all cases by 1month after treatment (IVA injection) and remained stable until the examination at the third month, she said that the median best corrected visual acuity in her study improved from 20/200 at baseline to 20/100 at 1month and 20/50 at 3months. They observed decreased neurosensory retinal and retinal pigment epithelial detachment on OCT. The retinal thickness decreased from 375.83±92.34μm to 252.5±58.28μm at 1month and 223±69.5μm at 3months post-treatment. There was a decrease in fluorescein leakage and improved choroidal hyperpermeability seen on indocyanine green angiography. Two eyes (1/3 of patients) required a second injection of bevacizumab, Dr. Torres-Soriano commented. No adverse events developed. However, they used higher concentration dose of Avastin (2.5mg).
Also our results in agreement with (Seong et al., 2009), in which they reported in a series of 10 eyes of 10 patients with acute CSC that all patients showed resolution of neurosensory detachment promptly, and improvement in visual acuity and symptoms within 1month. At 6months the mean BCVA (log MAR) had improved from 0.32 to 0.04, which was statistically significant (P=0.007). No recurrence was observed during the 6months follow up.
Our results in agreement with another series of (Schaal et al., 2009), they studied 12 eyes with chronic CSC in which patients received 2±1 intravitreal injections of bevacizumab 2.5mg on average during a follow up of 24±14weeks. Mean BCVA increased by 2±2 lines; the change in BCVA (log MAR) was significant (P<0.02). Mean central retinal thickness decreased significantly over follow up (P<0.05), with six patients (50%) showing complete resolution of subretinal fluid. Also in agreement with (El-Batarny et al., 2008), in their study of effect of one injection 1.25mg dose of IVA in 10 cases of chronic and persistent CSC, they concluded that 90% of patients had visual improvement. Six eyes (60%) gained two or more lines in BCVA at last follow up. The mean log MAR of BCVA improved from 0.50±0.25 at baseline to 0.11±0.16 at last follow up (P=0.003). Complete resolution was achieved in 60% of cases and CMT improved from 483.6±73μm (range, 632–412) at baseline to 274.6±104μm (range, 440–162) at last follow up with statistically significant difference (P=0.001).
Our results conclude that anatomic and functional improvement following intravitreal Avastin injections suggest that vascular endothelial growth factor (VEGF) may be involved in fluid leakage in patients with CSC. This is consistent with the previous studies that found that Avastin was effective in the treatment of different forms of CSC and resolution of subretinal fluid due to decreased choroidal hyperpermeability after IVA injection which it was proven to be markedly increased in cases of CSC. The results suggest a possible role for anti-VEGF agents in the treatment of CSC. Further evaluation of intravitreal Avastin for CSC patients in controlled randomized large number of patients with longer follow up period are necessary to confirm the efficacy and safety of bevacizumab and to determine the ideal protocol for this new promising treatment.
References
- Indocyanine green angiographic findings in central serous chorioretinopathy. Eye. 1995;9:324–332
- Photodynamic therapy for chronic central serous chorioretinopathy. Retina. 2003;23:752–763
- Corticosteroids and central serous chorioretinopathy. Ophthalmology. 2002;109(10):1834–1837
- Choroidal vascular remodeling in central serous chorioretinopathy after indocyanine green guided photodynamic therapy with verteporfin: a novel treatment at primary disease level. Br. J. Ophthalmol. 2003;87:1453–1458
- . Multifocal electroretinogram abnormalities persist following resolution of central serous chorioretinopathy. Arch. Ophthalmol. 2000;118:1211–1215
- Central serous chorioretinopathy and Helicobacter pylori. Eur. J. Ophthalmol. 2006;16(2):274–278
- Role of intravitreal bevacizumab (Avastin) in the management of chronic and recurrent central serous chorioretinopathy (CSC). Bull. Ophthalmol. Soc. Egypt. 2008;101(4):451–458
- Pigment epithelium decompensation, clinical features and natural course. Ophthalmology. 1984;91:1544–1548
- Factors associated with reduced visual acuity during long-term follow up of patients with idiopathic central serous chorioretinopathy. Retina. 2002;22:19–24
- Severe retinal thermal injury after indocyanine green-mediated photothrombosis for central serous chorioretinopathy. Am. J. Ophthalmol. 2007;143(5):887–889
- Intravitreal bevacizumab for treatment of chronic central serous chorioretinopathy. Eur. J. Ophthalmol. 2009;19(4):613–617
- Intravitreal bevacizumab to treat acute central serous chorioretinopathy: short-term effect. Ophthalmologica. 2009;223(5):343–347
- . Role of vascular endothelial growth factor in ocular angiogenesis. Ophthalmol. Clin. North Am. 2006;19(3):335–344
- . Central serous chorioretinopathy half dose verteporfin PDT for central serous chorioretinopathy. Br. J. Ophthalmol. 2006;90:805–806(editrorial)
- Sympathetic–parasympathetic activity and reactivity in central serous chorioretinopathy: a case-control study. Invest. Ophthalmol. Vis. Sci. 2006;47(8):3474–3478
- A pilot study of intravitreal bevacizumab for the treatment of central serous chorioretinopathy (case reports). Graefe’s Arch. Clin. Exp. Ophthalmol. 2008;246(9):1235–1239
- . Retinal atrophy in idiopathic central serous chorioretinopathy. Am. J. Ophthalmol. 2002;133:787–793
PII: S1319-4534(10)00045-7
doi:10.1016/j.sjopt.2010.03.002
© 2010 Published by Elsevier Inc.
