Macular Thickness and Volume Assessed by Spectral-Domain Optical Coherence Tomography in Patients With Coronavirus Disease 2019: A Case-Control Study

Purpose: To quantify the retinal thickness and volume using spectral-domain optical coherence tomography (SD-OCT) analysis in the macular region of patients with Coronavirus Disease 2019 (COVID-19). Methods: In a comparative cross-sectional, observational study, patients recovered from COVID-19 were included. All included subjects had a reverse transcription-polymerase chain reaction (RT-PCR) conrmed diagnosis of COVID-19. Macular SD-OCT was performed at least two weeks after recovery from systemic COVID-19. Inner, outer and full retinal thicknesses and volumes were measured in COVID-19 recovered patients versus age-matched normal controls. Results: Twenty-ve patients (11 male) with a mean age of 36.4 ± 11 years and 60 healthy controls (31 males) with a mean age of 39.3 ± 7.7 years were enrolled in the study. There was no statistically signicant difference in the retinal thickness or volume measures between the two groups. However, the thickness in the case group was minimally more than the controls. Conclusion: Retinal thickness in COVID-19 patients may be higher than healthy subjects. Comprehensive ocular examination with special focus on posterior segment manifestations should be considered in these patients.

Since the neurological manifestations of the SARS-CoV-2 have not been adequately studied yet, the casualty or coincidence of these ndings with COVID-19 in not clear. Usually, critically ill patients have shown a greater risk of developing neurological symptoms (7,8). As the retina and optic nerve are extensions of the central nervous system, evaluation of the retinal changes may help to identify the CNS changes as well. Indeed, it has been demonstrated that the SD-OCT is an effective diagnostic modality to detect many CNS pathologies (9,10).
The most common and easily identi able ocular manifestations of the SARS-CoV-2 have been reported in the ocular surface (e.g. conjunctival hyperemia, chemosis, and epiphora) (11)(12)(13)(14). However, there is a paucity of data on other ocular ndings in these patients. In this study, we aimed to evaluate the total, inner, and outer retinal thickness and retinal volume in the macula area of a cohort of patients with a con rmed diagnosis of COVID-19 and compare them with a control group.

Study Participants
A comparative observational study was conducted in Imam Reza Hospital, the referral center devoted to COVID-19 patients in Northeast of Iran. All included patients had a de nite diagnosis of COVID-19, con rmed by a positive real-time, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) test result of nasopharyngeal swab sample and had at least a two-week recovery period. A control group was retrospectively selected from participants in the ongoing PERSIAN cohort study in the Mashhad University of Medical Sciences (15). The individuals in the control group were examined before January 2020, one month before the rst report of a con rmed case of COVID-19 in the region. Two ophthalmologists (MA, MRA) overviewed the charts and SD-OCT images of the control group to ensure there were no abnormalities. Patients with a past medical history of diabetes mellitus, systemic hypertension, or known vascular or neurologic disorders, and those with a history of glaucoma or any intraocular surgeries were excluded from the study in both groups.

Image Acquisition
Macular area of the eligible subjects was imaged using an SD-OCT machine (AngioVue, Optovue RTVue The normal distribution of variables was evaluated through the Shapiro-Wilk test and normality plots. Based on the distribution, the independent-samples T test or Mann-Whitney U test was used to compare thickness measurements between the independent samples (control and case groups). For all tests, a pvalue less than or equal to 0.05 was considered as statistically signi cant.

Ethical Considerations
The study protocol adhered to the tenets of the 1964 Declaration of Helsinki and its later amendment. All subjects participated in the study voluntarily at their convenience and provided written informed consent at the enrolment. The study protocol was approved by the Medical Ethics Committee at the Mashhad University of Medical Sciences, Mashhad, Iran (IR.MUMS.REC.1399.104).

Results
Twenty-ve patients (11 male) with a mean age of 36.4 ± 11 years and 60 healthy controls (31 male) with a mean age of 39.3 ± 7.7 years were enrolled in the study. Age (P = 0.263) and gender (P = 0.635) were not signi cantly different between the two groups. Mean SSI was 75.7 ± 6.8 in the COVID-19 cases and 72.3 ± 8.3 in the normal controls (P = 0. 181).

Discussion
In the present study, we used SD-OCT to compare inner-, outer-, and full-retinal thickness and volume of the recovered COVID-19 patients with healthy control subjects. We found unremarkable incremental patterns in macular retinal thickness and volume in patients with a history of COVID-19. Although we failed to reach statistical signi cance, it seems that there is a trend toward retinal thickening in almost all sectors and different retinal layers of patients with COVID-19.
Recently, Marinho and colleagues reported hyperre ective bands in inner retinal layers in patients recovered from COVID-19 (4). Beside SD-OCT ndings, they have also reported ne Cotton-Wool Spots (CWS) and microhemorrhages along the retinal arcades in four patients, suggesting an in ammatory or ischemic process. However, they did not report the detailed quantitative measures of the retinal thickness parameters. Moreover, they had no control group and their sample size was limited. In a later editorial on their correspondence, Vavvas et al. raised some concerns on the interpretation of the OCT and fundus ndings in COVID-19 patients (5). They suggested that as CWS were very subtle and could be due to some comorbid conditions, the results provided by Marinho et al. should be interpreted with caution. In this study, we reported the detailed SD-OCT quantitative data of the retinal thickness in patients with a recent history of COVID-19 and compared our data with a healthy control group. Interestingly, we observed unremarkable changes in retinal thickness which may be in line with the theory suggested by Vavvas et al (5).
SARS-CoV-2 replication will be initiated after binding to epithelial cells in the nasopharynx and the nasal cavity. The virus propagates and migrates down the respiratory tract and triggers an innate immune response. ACE2 has been found as the main receptor for SARS-CoV-2 (16). ACE2 is an enzyme placed in cell membranes of type II alveolar cells of the lung, enterocytes of the small intestine, arterial and venous endothelial cells, and arterial smooth muscle cells in most organs (17). ACE2 counterbalances the activity of the ACE by reducing the amount of angiotensin-II and increasing angiotensin (1-7) (18). ACE2 has been found in human, rodent, and porcine retina. Moreover, ACE has been reported to present in the choroid and different cell types of the retina, including Müller cells, ganglion cells, retinal vascular endothelial cells, and photoreceptor cells (3). Hence, retina and choroid are well anticipated to be potential targets of SARS-CoV-2 infection (19).
In addition, subtle increasing in the thickness of retinal layers may be related to the minimal extra-cellular or intra-cellular uid accumulation. Vinores et al. performed an experimental study on murine coronavirus infection in susceptible mice (BALB/c) and found that blood-retinal barrier breakdown occurs in the early phase of infection, which coincide with the onset of in ammation and indicates that blood-retinal barrier breakdown is primarily due to in ammation rather than to retinal cell destruction (20). On the other hand, apoptosis plays an important role in many ocular pathologies, such as glaucoma, retinitis pigmentosa, and diabetic retinopathy (21). Wang et al. showed that murine coronavirus induced a biphasic retinal disease in adult mice (22). The early phase was associated with in ammation, including retinal vasculitis and viral replication and the late phase was associated with retinal degeneration. Based on these pieces of evidence, we hypothesize that retinal thickening in COVID-19 patients may happen in two phases: an initial in ammatory phase with increased vascular permeability followed by a late apoptotic phase with in ltration of the retina by in ammatory cells. Although there is no direct evidence which associate the virus with retinal changes, as the virus has been found in retinal autopsy samples and Our study had some limitations. First, we could not obtain OCT images during the acute phase of the disease. Second, our study had a relatively small sample size which might be the reason for insigni cance ndings. Third, since the control individuals were selected retrospectively from a large sample of an eligible cohort, selection bias might have occurred during the identi cation of the control arm. However, the mean thickness measures in our control group showed negligible difference with the previously published study in Iran which might be due to different measuring devices and sample ethnicity (23). A larger-scale study during the acute phase of the disease, followed by repeated exams at xed intervals, would provide valuable information about the impact of COVID-19 on the retina.

Conclusion
This study shows that the patients with COVID-19 developed unremarkable thickening, mainly in parafoveal and perifoveal temporal and superior quadrants of the macula. The ndings of the current study, either primarily or secondary to other causes, denote the retina could be involved in the patients with COVID-19 and highlight the necessity of comprehensive ophthalmic examinations in these patients. Consent for publication: As our study does not contain data from any individual person, so consent for publication is not applicable Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests