key: cord-0753056-p4n5acp7
authors: Cennamo, Gilda; Reibaldi, Michele; Montorio, Daniela; D'Andrea, Luca; Fallico, Matteo; Triassi, Maria
title: Optical coherence tomography angiography features in post COVID-19 pneumonia patients: a pilot study
date: 2021-03-27
journal: Am J Ophthalmol
DOI: 10.1016/j.ajo.2021.03.015
sha: 38dd01f078b2cd522cf818f4920b806f073e6a56
doc_id: 753056
cord_uid: p4n5acp7

PURPOSE: : To investigate retinal vessel density changes in macular and papillary regions in post SARS-CoV-2 pneumonia patients by means of optical coherence tomography angiography (OCTA). DESIGN: : Prospective, observational, cohort study. METHODS: : Forty eyes of 40 patients (mean age 49.7 ± 12.6) post SARS-CoV-2 infection and 40 healthy subjects were enrolled in this study. Covid-19 patients had to be fully recovered from Covid-19 pneumonia and were evaluated 6 months after Covid-19 infection. The primary outcome was the results of the OCTA study of the following vascular structures: vessel density (VD) in the retinal superficial capillary plexus (SCP), deep capillary plexus (DCP), radial peripapillary capillaries (RPC), compared to those of controls. We also evaluated the structural spectral domain (SD)-OCT parameters: ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL). RESULTS: : The patients showed a significant reduction in VD of the SCP in whole image and in the DCP in all sectors compared to healthy subjects (p<0.05). Covid-19 patients featured a reduced VD of the RPC compared to controls (p<0.001). No difference was found in the GCC, while the RNFL was reduced in the COVID-19 group compared to controls (p=0.012). Significant correlations were found between the RNFL and VD of the SCP, DCP, RPC and FAZ area in the Covid-19 group (p<0.05). CONCLUSION: : OCTA showed retinal vascular changes in subjects fully recovered from Covid-19 pneumonia. These findings could be a consequence of a thrombotic microangiopathy that affected retinal structures as well as other systemic organs. OCTA could represent a valid, non-invasive biomarker of early vascular dysfunction after SARS-CoV-2 infection.

Since December 2019, the SARS-CoV-2 outbreak has been a dramatic issue all over the world. On March the 11 th 2020, the WHO declared a pandemic. 1 All countries have been tremendously affected and all healthcare systems have been overwhelmed by this calamity. To date, no effective therapy has been developed and there is no clue as to whether a future vaccine will be able to stop it. 2 This infection can be completely asymptomatic, or it can involve several organs and tissues, eyes included. A hypercoagulable state, leading to thromboembolic events and disseminated intravascular coagulation, has been observed in many critical patients. 3, 4 Recent research demonstrated diffuse endothelial damage that causes ischemic injury to different body districts. Such an impairment of the microcirculatory system may lead to functional disorders in multiple organs. 5, 6 Ocular implications have not been fully studied. Non-specific retinal signs, such as microhemorrhages, vein dilatation, cotton-wool spots, and flame-shaped hemorrhages, have been reported in many recent studies. However, it was not possible to clearly establish whether these signs were secondary to COVID-19 or just incidental findings, given the high presence of comorbidities in the general population. [7] [8] [9] Optical coherence tomography angiography (OCTA), a new non-invasive imaging technique, may provide qualitative and quantitative features of retinal and choroidal vascularization and could monitor the changes of vascular perfusion in patients with Covid-19. 10, 11 The aim of this pilot study was to evaluate retinal vessel density (VD) in patients who fully recovered from Covid-19 pneumonia, and to compare these findings with healthy controls.

The present study was a prospective, observational, cohort study. The study protocol was registered on clinicaltrial.gov (NCT04601012). The study adhered to the tenets of the Declaration of Helsinki and was approved by the local Institutional Review Board. Written informed consent was obtained from all subjects enrolled in the study.

Consecutive patients who had been admitted to Hospital with Covid-19 pneumonia and had fully recovered from the infection were referred after 6 months from discharge to the Eye Clinic of the University of Naples "Federico II" in October 2020, and were assessed for eligibility. The following inclusion criteria had to be satisfied to be enrolled: a) history of hospital admission for Covid-19 pneumonia, classified as moderate illness, not requiring supplemental oxygen; b) full recovery; c) 2 consecutive upper respiratory tract samples negative for viral nucleid acid. Moderate illness was defined as evidence of disease affecting the lower respiratory tract with an SpO2 ≥ 94%, not requiring administration of supplemental oxygen. 12 Exclusion criteria were congenital eye disease, high myopia and high hyperopia (greater than 6 diopters), retinal vascular diseases, macular diseases, previous ocular surgery except uneventful cataract surgery, history of other ocular disorders, or significant lens opacity to avoid low-quality OCT-A images. All subjects with a history of stroke, blood disorders, diabetes, uncontrolled hypertension and neurodegenerative disease were also excluded. Each patient enrolled in the Covid-19 group was age-and gender-matched with a healthy control. Each subject underwent a complete ocular assessment including best corrected visual acuity measurement (BCVA), slit-lamp biomicroscopy, Goldmann applanation tonometry, dilated fundus examination. Snellen BCVA measurement was based on the Early Treatment Diabetic Retinopathy Study (ETDRS) charts (converted into logMAR for statistical analysis). Spectral domainoptical coherence tomography (SD-OCT) and OCTA were performed by two independent observers (GC, DM) that carefully reviewed the OCTA and SD-OCT scans to confirm accurate retinal layer segmentation.

Only one eye for each participant was randomly selected and included in the analysis.

The primary outcome of this study was the vessel density of macular and papillary regions on OCTA in the Covid-19 group, compared with the control group. Foveal avascular zone, sd-OCT parameters, such as ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL), were considered as secondary outcome measures, as well as clinical variables, including BCVA and retinal findings.

All the patients were examined using spectral domain-OCT (SD-OCT) (software RTVue XR version 2017.1.0.151, Optovue Inc., Fremont, CA, USA). The optic nerve head (ONH) analysis measures the disc area, the rim area, and the cup-to-disc ratio and was used to assess the RNFL thickness, calculated along a 3.45-mm diameter circle around the optic disc. The GCC thickness was obtained from a 7 × 7 mm grid of the macula centered 1-mm temporal to the fovea. The GCC thickness is the distance from the internal limiting membrane to the outer boundary of the inner plexiform layer. 13

All the subjects underwent OCTA (Optovue Angiovue System, software ReVue XR version 2017.1.0.151, Optovue Inc., Fremont, CA, USA). It is based on a split-spectrum amplitude decorrelation algorithm (SSADA). 14 The OCTA analysis divided the macular region into whole image, fovea and parafovea in each vascular network of the retina, according to the ETDRS classification of diabetic retinopathy. 15 The software (AngioAnalytic™) automatically calculated the vessel density in different retinal vascular networks: Superficial Capillary Plexus (SCP) and Deep Capillary Plexus (DCP) in a 6x6 mm quadrat scan centered on the fovea. Moreover, the software automatically calculated the Foveal Avascular Zone (FAZ) area in the full retinal plexus. 16 The VD of the Radial Peripapillary Capillary plexus (RPC), analyzing the whole papillary region, inside the disc and peripapillary region with an area scan of 4.5 x 4.5 mm, was automatically calculated by the Angio Vue disc mode. 17 The OCTA device included the 3D Projection Artifact Removal (PAR) algorithm to remove projection artifacts, for improving depth resolution on OCTA signal and then distinguishing vascular plexus-specific features. [18] [19] [20] Each OCTA scan underwent Automatic Scan Quality (1~10), values >=6 were accepted. OCTA images with a Signal Strength Index (SSI) less than 80 and residual motion artifacts were excluded from the analysis.

Statistical analysis was performed with the Statistical Package for Social Sciences (Version 25 for Windows; SPSS Inc, Chicago, Ill, USA). The Chi-squared test was used to determine differences in terms of sex. Student's t-test analysis for independent samples was used to compare structural SD-OCT and OCTA parameters between patients and controls. The multiple linear regression model was used to evaluate the relationship between OCT and OCTA parameters in the post Covid-19 group. The agreement between two observers in the measurement of SD-OCT and OCTA parameters was assessed using the intraclass correlation coefficient. A p value of < 0.05 was considered statistically significant.

A total of 40 eyes of 40 patients were included in the Covid-19 group (mean age 49.7 ± 12.6; 11 females and 29 males) and a total of 40 eyes of 40 age-and gender-matched healthy subjects in the control group (mean age 48.6 ± 12.2). Demographic and ocular characteristics of enrolled patients are reported in Table 1 . Mean BCVA was 0.06 ± 0.06 logMAR (Snellen 20/23) and 0.05 ± 0.05 logMAR (Snellen 20/22) in the Covid-19 group and the control group, respectively. All patients included in the Covid-19 group presented unremarkable ocular examination on the slit lamp as well as normal fundus examination. None of them complained of eye symptoms at the time of enrollment and no participant had a history of previous eye symptoms during hospital admission for Covid-19 pneumonia. All patients were phakic in both the Covid-19 and the control group. Recovery time from SARS-CoV-2 infection, confirmed by two consecutive negative oropharyngeal swabs, was 4.1 ± 1.3 months. There is no significant correlation between OCTA parameters and recovery time after SARS-CoV-2 infection (p=0.732).

On OCTA imaging, SSI value was comparable between the two groups (p=0.921). SCP vascular density was decreased in the COVID-19 group compared to the control group only in the whole image (p=0.038). DCP vascular density showed a significant reduction in all macular sectors in the COVID-19 group compared to the control group (p=0.029, p=0.016 and p=0.027 in whole image, parafovea and fovea, respectively). A significant reduction of RCP vessel density in the whole image was found in the COVID-19 group compared to the control group (p<0.001) (Table 2, Figure 1 ). The FAZ area did not show any significant change between the two study groups (p>0.05) (Table 2, Figure 1 ). The structural SD-OCT showed no significant difference in GCC average (p=0.309), while RNFL average was decreased (p=0.012) in the COVID-19 group compared to the control group (Table 2, Figure 1 ).

The agreement between two observers for measuring the SD-OCT and OCTA parameters was excellent, with an intraclass correlation coefficient >0.8 (Table 3) Multiple regression analysis revealed in all Covid-19 patients a significant relationship between reduced RNFL average thickness and impaired OCTA parameters (r=0.818, p=0.001), in particular with SCP parafovea (p=0.004), DPC whole image (p=0.006), DCP parafovea (p=0.002), RPC whole image (p=0.001), RPC inside (p=0.012) and FAZ area (p=0.008). No significant relationship was found between GCC average thickness and OCTA parameters (r=0.712, p=0.057) ( Table 4) .

To the best of our knowledge, this is the first report that investigated macular and peripapillary vessel density changes using OCTA in subjects after the Covid-19 infection. Our results show a significantly altered retinal vascular density in post Covid-19 subjects compared with healthy controls: DCP vessel density was reduced in all macular regions, while SCP and RCP vessel density was reduced only in the whole image. These findings could be explained by the multiple pathogenic mechanisms linked to the SARS-CoV-2 infection, including thrombo-inflammatory microangiopathy and angiotensin-converting enzyme (ACE) 2 disruption. 6, 21 Complement-mediated thrombotic microangiopathy (TMA) has been assumed to be one of the main factors involved in Covid-19 related microvascular damage. 22 Complement activation plays a central role in the pathophysiology of TMA determining platelet activation, leukocyte recruitment, endothelial cell dysfunction and coagulation. 23, 24 Complement cascade activation is, in turn, a response to an endothelial injury secondary to local renin-angiotensin system disruption. 6, 21 Endothelial cells express high levels of angiotensin-converting enzyme 2 (ACE 2) receptors, which are used by SARS-CoV-2 to gain entry into the cell that is then disrupted. 25 Endothelial damage and subsequent thrombo-inflammatory microangiopathy lead to a hypercoagulative state that may explain the microvascular occlusion and the consequent multi-organ failure that characterizes advanced disease. 21, 26 Although severe respiratory complications are the main clinical features, Covid-19associated coagulopathy predisposes to a very wide spectrum of thromboembolic events, including pulmonary embolism, large-vessel ischemic strokes, venous thrombosis, renal failure, and cardiomyopathy, which can culminate in multiple organ dysfunction. 21, 27, 28 It should be noted that immunohistochemical studies conducted on the human eye reported that also the ciliary body, choroid, retina and retinal pigment epithelium showed significant levels of ACE 2 receptors. 29 Therefore, SARS-CoV-2 could cause microvascular damage to retinal and choroidal vessels. 9 Several retinal findings have been reported in Covid-19 patients, such as cotton-wool exudates, retinal flame-shaped hemorrhages, central retinal artery occlusion, and sectorial retinal pallor. All these could be considered signs of retinal vascular impairment following thrombotic complications. [7] [8] [9] 30, 31 Hitherto, only one report conducted by Savastano et al. used OCTA imaging in Covid-19 patients. 32 The authors demonstrated a reduction of perfusion density of the radial peripapillary capillary plexus in Covid-19 patients compared to healthy controls after one month from infection. Our results on RCP are in line with those of Savastano et al. 32 However, OCTA imaging focused mainly on the study of RCP, with no information on other regions or plexuses. Furthermore, the present study has shed light on vascular changes affecting capillary plexuses of the macula area as well. Our analysis also revealed a significant relationship between RNFL and OCTA parameters, which could be explained by the anatomical localization of the RCP in the peripapillary RNFL. 33 Covid-19 related thrombotic microangiopathy could have caused vascular perfusion damage to the SCP and RPC, leading to interference in axoplasmic flow and subsequent retinal structural loss. The anastomosis that interconnect the SCP with the DCP may explain the possible correlation between this plexus and the RNFL.

Interestingly, our findings showed no significant change in GCC thickness. Ganglion cell layer is expected to get reduced when there is a thinning of RNFL, since the latter one consists mainly of the ganglion cell axons. Probably, the vessel density of SCP was not compromised enough to cause such a reduction of metabolic support to GCC that would have determined an anatomic thinning of this layer.

OCTA highlighted a much more significant impairment of the DCP than the SCP in Covid-19 patients compared to controls, as also occurs in diabetic retinopathy and other systemic vasculopathies. 34, 35 The vascular structure of the deep plexus is characterized by a fine capillary network that makes it more vulnerable to thrombotic events compared to the greater vascular calibre of the SCP. 36 Of note, our OCTA findings showed that the perfusion loss was diffuse, with no specific pattern. Similarly, no specific pattern was demonstrated in RNFL thinning, which was a diffuse reduction of the whole area. There is no sectorial co-localization but a significantly correlation between damages in OCT and OCTA parameters.

There was no pattern of retinal structural and perfusion loss and no sectorial colocalization of damages in OCT and OCTA parameters All patients enrolled in the Covid-19 group of this study had no visual acuity reduction, no visual symptoms and their ocular examination proved unremarkable. They also had no history of any eye symptoms during their admission. The presence of retinal microvascular changes on OCTA imaging in otherwise healthy and asymptomatic eyes has a great scientific relevance. This might support the hypothesis of widespread microvascular damage that could be clinically silent. Idilman et al. found lung and kidney perfusion deficits in patients affected by mild to moderate Covid-19. 37 Dual-energy computed tomography angiography showed lung perfusion deficits in 26% of cases, most of which had no detectable emboli in pulmonary arteries and did not overlap with areas of consolidation or ground glass opacity. Kidney perfusion deficits were demonstrated in 50% of cases, in the absence of major kidney dysfunction. 37 Similarly, retinal microvascular changes did not cause clinical symptoms. It would be useful to know whether these microvascular deficits are associated with alterations on microperimetry and electrodiagnostic testing. On the other hand, a study conducted by Bussani et al. reported a persistence of virus-infected cells in lung pneumocytes and endothelial cells several weeks from COVID-19 diagnosis. 38 These findings could explain the retinal vascular changes as long-term consequences of SARS-CoV-2 infection. More importantly, long-term follow-up is needed to see whether this subclinical microvascular impairment could be responsible for the development of ischemic diseases or a choroidal neovascular membrane. Further studies are warranted for such purposes.

The present trial has some limitations. First, the sample size was relatively small. Second, no data on vascular changes at the time of acute infection were available. There is no information on long-term follow-up. However, this is a novel coronavirus disease and more time is needed to get longer follow-ups. No fluorescein angiogram was performed to investigate the retinal periphery. However, the purpose of the study was to analyze macular and peripapillary regions by using a dyeless methodology. Further longitudinal studies are needed to evaluate the correlation between the OCTA parameters and both the onset and the duration of SARS-CoV-2 infection.

In conclusion, OCTA detected retinal microvascular changes following SARS-CoV-2 infection, helping to highlight the presence of microvascular damage in clinically asymptomatic eyes. In this clinical scenario, where physicians and scientists have been puzzled as to how deal with an utterly new viral infection, new insights in pathogenetic mechanisms could be meaningfully appreciated. This non-invasive imaging technique could represent a valid biomarker of systemic vascular dysfunction as well. Longitudinal studies on larger cohorts are needed to detect the possible progression of retinal vascular alterations on long-term follow-up.

No financial disclosures.

We wish to thank the Scientific Bureau of the University of Catania for language support.

The patients post COVID-19 show a significant decrease in vessel density in macular and papillary regions at optical coherence tomography angiography (OCTA) and an impairment of RNFL thickness respect to healthy subjects. Statistically significant correlations were found between structural spectral domain OCT and OCTA parameters. OCTA allows to detect the signs of retinal thrombotic microangiopathy that could reflect the systemic vascular impairment occuring in multiorgan dysfunction. OCTA could represent a valid biomarker of systemic vascular disfunction after SARS-CoV-2 infection. 

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SCP: superficial capillary plexus; DCP: deep capillary plexus; RPC: radial peripapillary capillary plexus; FAZ: foveal avascu lar zone

GCC: ganglion cell comples; RNFL: retinal nerve fiber layer