How does Covid-19 affect the choroidal structures at the early post-infectious period?

Abstract

Purpose

To evaluate choroidal thickness (CT) and choroidal vascularity index (CVI) in patients recovered from COVID-19 using enhanced depth imaging optical coherence tomography in the early postinfectious period.

Methods

Sixty-five patients recovered from COVID-19 and 72 healthy subjects were included in the study. A full ophthalmic examination including best-corrected visual acuity (BCVA), slit lamp biomicroscopy, and dilated fundus examination was performed. CT was measured at 3 points as follows: subfoveal, 1000 μm nasal and temporal to the fovea. The total choroidal area (TCA), luminal area (LA), stromal area (SA), and CVI were measured with Image-J.

Results

The mean age was 39.09 ± 11.27 years in the COVID-19 group and 39.61 ± 11.43 years in the control group. The mean time from the first positive RT-PCR was 49.54 ± 26.82 days (range 18–120) in the COVID-19 group. No statistically significant difference was detected between the groups with regard to axial length, spherical equivalent, and BCVA (all P > 0.05). CT was found to be lower in the COVID-19 group compared to the control group in all quadrants, but this difference was not significant (all P > 0.05). The mean TCA, LA, and CVI were statistically significantly reduced in the COVID-19 group (all P < 0.001); however, SA showed no statistically significant difference (P = 0.064).

Conclusions

In asymptomatic or mild COVID-19, CVI and LA decrease significantly, while CT thins in the early postinfectious period but not significantly.

Introduction

The SARS-CoV-2, is enveloped, positive single-stranded RNA viruses belonging to the genus Beta coronavirus. Declared a pandemic by the WHO on the 11th of March 2020, coronavirus disease 2019 (COVID-19) has become a global health threat, still continuing to show its impact all over the world. The respiratory tract is the first site of COVID-19. SARS-COV-2 spike proteins bind to angiotensin-converting enzyme 2 (ACE2) receptors on the surface of type II alveolar cells. The type II transmembrane serine protease binds to the ACE2 receptor and makes it cleaved and activated, and then viral entry into the cell takes place [1]. Additionally, it is reported that CD147 known as basigin or EMMPRIN (extracellular matrix metalloproteinase induce) is the other receptor bound by the virus [2]. Shortly after the onset of the pandemic, it was seen that COVID-19 also affected other organs apart from the lungs, such as the intestine, heart, kidney, and eye, which express ACE2. The presence of ACE2 and its receptors in Müller cells, ganglion cells, retinal vessel endothelium, photoreceptors, choroid tissue, ciliary body and vitreous is known [3]. CD 147 has also been demonstrated immunohistochemically in the neural retina, retinal pigment epithelium (RPE), optic nerve head, and vitreous gel [4]. Due to the presence of ACE2 and CD147 in various ocular structures, eye tissue becomes a direct target for SARS-CoV-2. The presence of SARS-CoV-2 ribonucleic acid in the retina and optic nerve has also been demonstrated in biopsies from patients who died of COVID-19 [5]. One of the most important target tissues of COVID-19 is the vascular endothelium and vascular smooth muscle cells, which express ACE2 [6]. Choroid is a densely vascularized tissue. Due to its specialized structure, the choroid has the highest blood flow ratio of all human anatomical tissues considering unit per weight and becomes one of the target tissues affected by SARS-CoV-2. Therefore, this study aimed to evaluate choroidal thickness (CT) and choroidal vascularity index (CVI) in the early postinfectious period in patients recovered from COVID-19.

Materials and methods

The study followed the tenets of the Declaration of Helsinki. Before the study was initiated, ethical board approval was obtained from the ethics committee of our university. Written informed consents were obtained from all participants.

A total of 137 right eyes of 137 participants were included in this study. The COVID-19 group consisted of 65 patients who were asymptomatic or non-hospitalized with confirmed COVID-19 by a positive test result with RT-PCR. Patients admitted to the hospital or intensive care unit due to COVID-19 were excluded from the study. The patients used favipiravir, ascorbic acid, and paracetamol for the treatment of COVID-19. None of the patients used anticoagulant or antiplatelet medication. The control group consisted of 72 age and sex-matched healthy individuals. COVID-19 and control group with pseudophakia/aphakia or history of any intraocular surgery and laser treatment, high intraocular pressure [(IOP) (> 21 mmHg)]/glaucoma, high myopia [an axial length (AL) ≥ 26 mm], more than ± 3 diopters spherical equivalent (SE), uveitis, pregnancy, diabetes mellitus, systemic hypertension, choroidal or retinal disorders were excluded from the study. Participants who did not have a history of fever, cough, myalgia, weakness, headache, rhinorrhea, diarrhea, sore throat, loss of taste-smell, contact with a COVID-19 patient and quarantine since the beginning of the pandemic constituted the healthy control group. In addition, retrospective RT-PCR results were scanned from the medical records of healthy subjects for the absence of positive results. All participants in both the COVID-19 and control groups were phakic and had best-corrected visual acuity (BCVA) greater than 8/10. All the participants had a detailed ophthalmic examination including manifest refraction, BCVA expressed in decimals according to the Snellen chart, IOP with applanation tonometer, slit-lamp biomicroscopy, dilated fundoscopy, and AL measurement. The AL was measured using AL-Scan (Nidek CO., Gamagori, Japan). The manifest refraction was measured with an auto kerato-refractometer (TOPCON KR-8900; Topcon Corporation, Tokyo, Japan). The SE refractive error was calculated by adding the sum of the sphere power with half of the cylinder power. Pupil dilation was induced by 2 doses of tropicamide 1% applied 5 minutes apart.

The spectral-domain optical coherence tomography (SD-OCT) was performed by the same experienced technician using the Spectralis (Heidelberg Engineering, Heidelberg, Germany) before dilation. All images were obtained between 9:00 and 12:00 a.m. for preventing diurnal variations. Enhanced Depth Imaging (EDI) mode was used to image the choroidal tissue. Choroidal thickness was measured from the outer border of the hyperreflective line (corresponding to the RPE) to the hyperreflective line of the inner sclera border at the fovea, 1000 μm nasal to the fovea, and 1000 μm temporal to the fovea. The CT measurement was obtained manually using the caliper by two experienced observers. Two masked and independent ophthalmologists (MBU, SC) measured CT to eliminate inter-observer variability. The mean value of the measurements was calculated. If more than a 15% difference was detected between the reports of the two ophthalmologists, these measurements were excluded from the study.

ImageJ software (version 1.8.0_77, Bethesda, MD, USA http://imagej.nih.gov/ij/) was used to calculate the CVI (Fig. 1 ). The images obtained with EDI-OCT were converted using the ImageJ software, and the scale was adjusted to 200μm. The image type was changed to 8 bits and an automated local threshold was applied using the Niblack method [7] to convert all pixels from red, green, and blue to black and white. The number of pixels was measured using the histogram module in which the total number of pixels could be measured using the count number. The following values were obtained: Total choroidal area (TCA) = Luminal area (LA) +  Stromal area (SA) and the CVI = LA÷TCA.

Figure 1.

A: Enhanced-depth optical coherence tomography image; B: The image was binarized using Niblack's auto-local threshold; C: The color threshold tool was used to select the dark pixels, representing the luminal area.

Statistical Analysis

Statistical analysis of the data was performed with the SPSS (SPSS Inc., Chicago, IL, USA) package program. The normal distribution of the data was tested with the Kolmogorov-Smirnov test. Descriptive statistics for normally distributed continuous data were reported with mean ± standard deviation (SD). Descriptive statistics for categorical variables were presented with frequencies and percentages. Proportion comparisons between research groups were performed with either the Chi² test or Fisher's exact test, depending on the sample size in the crosstab cells. The comparison of continuous data between the two independent groups was conducted with the student's t-test for normally distributed data and the Mann–Whitney U test for non-normally distributed data. Statistical significance level was accepted as P  < 0.05.

Results

The mean age of the 65 patients (39 female, 26 male) was 39.09 ± 11.27 years (range 18–61) in the COVID-19 group and that of the 72 healthy individuals (43 female, 29 male) was 39.6 ± 11.43 years (range 18–54). The mean time from the first positive RT-PCR was 49.54 ± 26.82 days (range 18–120) in the COVID-19 group. There were no significant differences between the COVID-19 and control groups in terms of age (P  = 0.974) and gender (P  = 0.740) distribution. Besides, no statistically significant difference was detected between the groups with regard to AL (P  = 0.774), SE (P  = 0.858), BCVA (P  = 0.971), and IOP (P  = 0.233). The demographic characteristics of the patients and control subjects are presented in Table 1 . Although it was not statistically significant, CT was found to be lower in the COVID-19 group compared to the control group at the subfoveal (P  = 0.366), nasal (P  = 0.465), and temporal (P  = 0.836) quadrants. Detailed analysis of CT at different locations between the COVID-19 and control groups are presented in Table 2 .

Table 1.

Comparison of ophthalmologic features and demographic characteristics of subjects amoung COVID-19 group and control group.

	COVID-19 (n = 65)	Control (n = 72)	P
Sex (F/M)	39 (47.6%)/26 (47.3%)	43 (52.4%)/29 (52.7%)	0.974a
Age (years)	41 (18–61) 39.09 ± 11.27	41 (18–54) 39.61 ± 11.43	0.740b
AL (mm)	23.32 ± 0.69	23.29 ± 0.51	0.774b
SE (D)	−0.22 ± 1.03	−0.21 ± 1.04	0.858b
IOP (mmHg)	15.22 ± 1.70	15.31 ± 2.77	0.233b
BCVA (Snellen)	0.96 ± 0.06	0.97 ± 0.06	0.971b

F: Female; M: Male; AL: Axial length; SE: Spherical equivalent; D: Diopters, IOP: Intraocular pressure; BCVA: Best corrected visual acuity.

^aChi² test with n(%).

^bMann–Whitney U test with mean ± standard deviation.

Table 2.

Comparison of choroidal thickness (μm) at the fovea, 1000 μm nasal to the fovea, and 1000 μm temporal to the fovea between COVID-19 and control group.

	COVID-19	Control	P
Choroidal thickness at nasal quadrant	279.8 ± 47.37	287.5 ± 54.09	0.465
Subfoveal choroidal thickness	330.57 ± 54.08	342.19 ± 51.63	0.366
Choroidal thickness at temporal quadrant	292.89 ± 56.91	300.39 ± 52.35	0.836

Mann–Whitney U test with mean ± standard deviation.

The mean TCA was reduced statistically significantly in the COVID-19 group (0.56 ± 0.16 mm2) compared to the control group (0.67 ± 0.10 mm²) (P  < 0.001). The mean CVI was decreased statistically significantly in the COVID-19 group (0.65 ± 0.03) compared to the control group (0.69 ± 0.03) (P  < 0.001). Besides, the mean LA was statistically significantly reduced in the COVID-19 group (P  < 0.001), and the mean SA showed no statistically significant difference (P  = 0.064). Choroidal structural characteristics of the COVID-19 and control group are shown in Table 3 .

Table 3.

Comparison of total choroidal area, luminal area, stromal area and choroidal vascularity index values amoung COVID-19 and control group.

	COVID-19	Control	P
TCA (mm2)	0.56 ± 0.16	0.67 ± 0.10	< 0.001
LA (mm2)	0.36 ± 0.09	0.46 ± 0.06	< 0.001
SA (mm2)	0.19 ± 0.06	0.21 ± 0.04	0.064
CVI (%)	0.65 ± 0.03	0.69 ± 0.03	< 0.001

Mann-Whitney U test with mean ± standard deviation. TCA: Total choroidal area; LA: Luminal area; SA: Stromal area; CVI: Choroidal vascularity index.

Discussion

In our study, subfoveal, nasal and temporal CT was found to be thinner in the COVID-19 group, yet the difference was not statistically significant. This study also evaluated the effect of COVID-19 on CT in the early post-infectious period. The period of enrollment of patients after the first RT-PCR positivity was 49.54 ± 26.82 days (range 18–120). The literature includes controversial results about the effect of COVID-19 on CT in the active disease period and during the recovery period.

Kocamış et al. found that the mean CT of moderate COVID-19 patients was thinner at subfoveal, temporal and nasal quadrants; but the difference was not statistically significant [8]. When the CT of COVID-19 patients was measured again 4 months after remission, the mean CT in all 3 quadrants was found to increase, but not significantly. However, it was observed that the choroid remained thinner than the control group. Hepokur et al. reported that the mean CT in subfoveal, nasal and temporal quadrants was significantly thinner in patients recovered from COVID-19 in the early post-infectious period (15 to 40 days after the onset of COVID-19) who were not hospitalized, did not use anticoagulant or antiaggregant therapy [9]. The authors also reported that the peripapillary CT was thinner in the early post-infectious period. It was observed that the CT of the same patients increased in all quadrants in the late post-infectious period (after 9 months) and did not make a significant difference with the control group. Interestingly, it was observed that the CT of the subfoveal and peripapillary of patients was still thinner in many quadrants than in the control group, even in the late post-infectious period. Erdem et al. found thinning of CT in all quadrants in the early post-infectious period (time elapsed since the diagnosis of COVID-19 was 53.18 ± 2.84 days) in patients who had recovered from COVID-19 [10]. Contrary to our study, Zor et al. found significant choroidal thickening in some quadrants in patients recovered from COVID-19 (within 2 months) who were not hospitalized and did not use antiplatelets [11]. Since the immune response is the peak on the 8th day in the COVID-19, the authors speculated that choroid would be even thicker in the early phases of the disease, and parallel to the decrease in the immune response, they speculated that the CT might have decreased after the peak. In the study conducted by Bayram et al. included patients with severe COVID-19 with an average age of 50.2 ± 7.4 years and reported that the subfoveal, nasal and temporal CT was significantly higher in the active disease before the treatment was started [12]. In the third month measurements, it was observed that the choroid was thinned and did not make a difference with the control group. The authors reported that severe edema developed in the choroidal stroma during the active disease, and the choroidal reflectivity of OCT echo was increased with a weak positive correlation of the concentration levels of acute-phase reactant.

The exact pathophysiological mechanism of COVID-19 is largely unknown. However, SARS-CoV-2 causes tissue damage through various pathways. In patients with severe COVID-19, histopathologically, the literature reports viral inclusion structures in the glomerular capillary endothelium and diffuse endotheliitis in the heart, lung, kidney, liver and gastrointestinal tract [13]. Healthy endothelium naturally expresses factors that induce vascular relaxation, increase blood flow, inhibit platelet aggregation and coagulation, and promote fibrinolysis while dysfunctional endothelium shifts the balance to vascular contraction and thrombus formation. SARS-CoV-2 directly attaches to endothelial cells, causing microvascular leakage, microvascular coagulation, excessive release of inflammatory cytokines, disruption of cell-cell contact, endothelial dysfunction associated with ischemia and thrombosis [14]. COVID-19 causes immune dysregulation and inappropriate proinflammatory cytokine response. The SARS-CoV-2 cytokine storm precipitates the onset of a systemic inflammatory response syndrome, resulting in the activation of the coagulation cascade that induces a hypercoagulable state and disseminated intravascular coagulation [15]. For these reasons, it is believed that many patients with COVID-19 may have thrombotic microangiopathy and venous or arterial thromboembolic complications [16]. The prevalence of thromboembolic events such as myocardial ischemia, deep vein thrombosis, acute cerebral infarction and pulmonary embolism increased during and after COVID-19.

Several optical coherence tomography angiography (OCTA) studies have shown a reduction in vessel density in COVID-19 patients, confirming the possibility of COVID-19 related retinal microvasculopathy. These perfusion deficiencies have even been described in asymptomatic individuals recovering from mild COVID-19 and without significant medical comorbidities [17], [18], [19], [20]. In addition, some retinal findings reflecting the existing thrombotic ischemic process in COVID-19 patients such as flame-shaped hemorrhages [21], dot blot hemorrhages [22], soft exudates [23], [24], [25], Roth spots [22], isolated cilioretinal artery occlusion [26], dilated veins [24], and papillophlebitis [16] have been reported clinically. These retinal changes can also be of a secondary nature due to the poor general condition of the affected patients. However, Sim et al. observed increased vascular tortuosity, soft exudates and retinal hemorrhages even in asymptomatic young patients with normal vital signs [25]. This strengthens the evidence that COVID-19 has thrombotic-ischemic effects in the posterior segment. Furthermore, analysis of the literature shows that COVID-19 is associated with an 8.86-fold retinal microvasculopathy prevalence [27].

Choroidal vascular involvement has been demonstrated by ICGA in patients with COVID-19. The study found that 68% of the eyes had hypocyanescent dark spots that continued throughout the entire angiographic sequence, 36% had intervortex shunts, and 18% had hemangioma-like lesions. In OCTA scans, choriocapillaris hypoperfusion was observed in regions matching these accessible ischemic hypocyanescent lesions [28]. Another study reported a significantly reduced vascular density in the choriocapillaris with OCTA in recovered patients [18]. The dropout of the choriocapillaris could increase vascular resistance, resulting in decreased blood flow in the choriocapillaris [29]. The increase in hypoperfusion and vascular resistance in the choriocapillaris and other choroidal vascular structures as a result of prothrombosis, inappropriate inflammation, endotheliitis and the resulting microvasculopathy may be related to the decrease in CT at the early post-infectious period in patients with COVID-19.

Additionally, ACE2 is a counterregulatory enzyme that degrades angiotensin II (Ang II) to angiotensin 1-7, which is a vasodilator with anti-oxidant and anti-inflammatory effects, thereby attenuating its effects on vasoconstriction, sodium retention, and fibrosis [14]. In COVID-19, ACE2 is down-regulated and the renin-angiotensin system is dysregulated, increasing Ang II production and effect. Angiotensin II mediates vasoconstriction, fibrosis, hypertrophy, and inflammation and thereby exacerbating the microangiopathy present in patients with COVID-19. COVID-19 may also activate the sympathetic system which is a compound of the autonomic nervous system through increased production of Ang II [30]. The autonomic nervous system exerts a modulatory effect on the immune system. While sympathetic activation is proinflammatory [31], [32], [33], parasympathetic vagal stimulation has anti-inflammatory [34], [35], [36] effects. In addition to systemic inflammation, secondary local inflammation in the choroidal tissue in COVID-19 may affect choroidal autonomic control. These sympathetic noradrenergic neurons terminate in blood vessels and cause vasoconstriction. In addition to microvasculopathy and vascular dysfunction, sympathetic activation may contribute to the reduction of CT by reducing blood flow in the choroid.

Choroid thickness is reported as an indicator of ocular and systemic health and may be affected in some ocular or systemic diseases. Agrawal et al. reported that CVI shows less variability and is less affected by physiological factors than CT, which means that CVI is a relatively stable index to evaluate changes in the choroid tissue [37]. It has been suggested that the dark areas as LA in binary images are vascular components that include both larger and smaller choroidal vessels [38], [39]. An increase in CVI reflects an increase either in the number of vessels or in the diameter of vessels within a designated area. In our study, while CVI and LA were significantly lower in the COVID-19 group, no significant change was observed in SA. These results may show that at least in the asymptomatic or mildly non-hospitalized patients in our study, COVID-19 may affect the choroidal vascular structure with a decrease in the number of vessels or narrowing in the vessel lumen due to systemic and local inflammation, endotheliitis, and prothrombotic state. In addition, a non-significant change in SA indicates that the choroidal stroma may be affected less than the choroidal vascular structure. Bayram et al. found the CVI lower and the SA/LA ratio higher in patients with severe COVID-19 [12]. The authors reported that this may be due to choroidal vessel wall thickening and narrowing of the lumen as a result of choroidal stromal edema and endothelial inflammation. They also reported that microthromboembolic complications contributed to this situation. Another study reported that TCA decreased in the early post-infectious period, but became nonsignificant in the late postinfectious period while CVI showed no significant change in the early-late post-infectious and control group. The authors speculated that structural recovering of the choroid occurred simultaneously in both the stroma and blood vessels as the CVI remained the same despite significant choroidal thickening in the late post-infectious period [9].

One of the strengths of the current study is that it evaluated only non-hospitalized outpatients with mild disease or asymptomatics rather than those with varying degrees of the disease because COVID-19 generally shows asymptomatic or mild clinical course (no pneumonia or mild pneumonia) in the population [40]. In addition, since patients with serious diseases or patients admitted in the intensive care unit were excluded from the study, the potential effects of systemic anti-inflammatory drugs, steroids, antithrombotics and even oxygen support used in the treatment were eliminated on the choroidal tissue. The major limitation of this study is that choroidal measurements can not be performed in the active disease because patients with COVID-19 are under home quarantine. Therefore, a comparison of the choroidal structure during active disease and recovery period could not be evaluated. In addition, the presence of pneumonia of the patients in the study population is unknown. The lack of simultaneous OCTA imaging in may be another limitation. Choroidal thickness measurement was performed with a manual caliper. However, this negativity was minimized by excluding the patient from the study if there was a 15% difference in CT between the results of the 2 observers. In addition, the healthy control group was constituted by evaluating the detailed anamnesis/symptom query, contact history with the COVID-19 patient and had no RT-PCR tests but only retrospective tests. Thus, although the possibility of asymptomatic COVID-19 patients in the healthy group is minimized, it cannot be excluded. This may be another limitation of the study.

Conclusion

In the present study, asymptomatic or mild COVID- 19 may affect the CT in the early post-infectious period due to inappropriate immune dysregulation, prothrombotic state, endotheliitis and microvasculopathy but not significantly. In addition, the significant reduction of CVI and LA may indicate that COVID-19 can affect the choroidal vascular structure even in asymptomatic or mild patients.

Funding

There are no any funds.

Disclosure of interest

The authors declare that they have no competing interest.

Ethical approval

All procedures performed in studies involving human participants were inn accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. IRB/ethics committee name, date and number: Hitit University Faculty of Medicine, 08/04/2021-405.

Informed consent

Informed consent was obtained from all individual participants included in the study.