Ocular surface manifestation of COVID-19 and tear film analysis

To evaluate the ocular manifestation in patients hospitalized with coronavirus disease 2019 (COVID-19) and to search for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in tears. This study was conducted in 29 hospitalized patients who were admitted to the COVID center at the Policlinic Hospital of the University of Messina, Italy. All patients underwent an ophthalmologic assessment comprising a Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaire, anterior segment, and the ocular surface examination of both eyes using a portable slit lamp. The Schirmer I test was performed, and the filter paper strip was used to search for the presence of SARS-CoV-2 on the ocular surface by real-time quantitative polymerase chain reaction (RT-qPCR). A total of 10 patients reported ocular symptoms; in particular, four reported eye burning, three reported foreign body sensation, and three reported tearing. Moreover, seven patients presented conjunctival hyperemia and/or chemosis, eleven patients presented blepharitis signs such as lid margin hyperemia and/or telangiectasia, crusted eyelashes, and meibomian orifices alterations. Tear analysis did not reveal the presence of SARS-CoV-2. Ocular symptoms are common in patients with COVID-19; although, tear analysis did not reveal the presence of SARS-CoV-2.


Scientific Reports
| (2020) 10:20178 | https://doi.org/10.1038/s41598-020-77194-9 www.nature.com/scientificreports/ Results of the ophthalmologic assessment. The mean value for the Schirmer I test was 9.2 ± 6.4 mm/5 min in the right eye and 9.3 ± 6.4 mm/5 min in the left eye. The mean Standardized Patient Evaluation of Eye Dryness (SPEED) questionnaire (composite) score was 2.3 ± 1.7. Ten patients described ocular symptoms following the onset of COVID-19; in particular, four reported eye burning, three reported foreign body sensation, and three reported tearing. In 17 patients, ocular findings were observed, such as conjunctival hyperemia, ocular secretion, lid margin hyperemia and/or telangiectasia, crusted eyelashes, and meibomian orifices alterations; however, only five patients who reported ocular symptoms actually presented with evident ocular manifestations at the ophthalmologic evaluation. Seven patients presented conjunctival hyperemia and or chemosis, eleven patients presented blepharitis signs.
Only one patient presented with conjunctivitis with moderate hyperemia, chemosis, and secretions (Table 2). No patients reported a loss of vision. Three patients reported a history of diabetic retinopathy, and one patient had glaucoma that was being treated with latanoprost eye drops in both eyes. Seven patients had previously undergone cataract surgery, all of whom reported ocular symptoms. No significant difference was observed in patients with and without previous eye disease regarding ocular symptom assessment (p = 0.98) ( Table 3).
Tear sample analysis results. In all evaluated tear samples, the control sample presented a C(t) between 24 and 27; however, no analyzed tear sample showed a positive result for SARS-CoV-2. The nasopharyngeal swab, evaluated on the same day as tear collection, was negative in four patients and positive in 25 patients.
Correlation between ophthalmologic findings and COVID-19. A significant correlation was found between conjunctival hyperemia and severity of the disease (ρ s = 0.55; p = 0.002), and between blepharitis-like findings and duration of the disease (ρ s = -0.54; p = 0.003). The composite SPEED score was significantly correlated with the presence of blepharitis findings (ρ s = 0.95 p; < 0.0001), but not with conjunctival findings (p = 0.11) ( Table 4).

Discussion
Numerous respiratory viruses, such as adenovirus, influenza virus, respiratory syncytial virus, coronavirus, and rhinovirus, exhibit an ocular tropism, which could be responsible for ocular infections 16 . Although previous studies have documented the presence of coronavirus in tears from patients with SARS-CoV infection, specific www.nature.com/scientificreports/ ocular involvement has not been described with either Middle-East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV [17][18][19][20] . Similar to other coronaviruses, the main route of transmission of SARS-CoV-2 is respiratory droplets and direct contact; however, the human ocular surface may represent a further exposure site. In fact, infectious droplets and bodily fluids can easily contaminate the corneal − conjunctival epithelium 21 . Furthermore, ocular tissue could be a potential site of viral replication due to the expression of ACE-2 receptors, which can supposedly bind SARS-CoV-2 to cause infection 22 .  www.nature.com/scientificreports/ The ocular surface is able to create a defensive barrier against several pathogens via the production of antimicrobial peptides such as immunoglobulins, lysozyme, and lactoferrin, as well as the presence of normal components of the bacterial flora [23][24][25] . Moreover, the mucin layer helps to avoid the binding of the virus to its epithelial receptors, potentially promoting its clearance through nasolacrimal drainage and acting as a gateway for the transfer of SARS-CoV-2 to the nose and respiratory mucosa 23,24 .
In the present study, we document a high prevalence of mild ocular symptoms reported by patients diagnosed with COVID-19. Furthermore, ocular assessment demonstrated the presence of mild conjunctival hyperemia, chemosis, and secretions, as well as the occurrence of eyelid margin hyperemia and other signs referable to blepharitis. On the other hand, ocular findings such as eyelid margin hyperemia and crusted eyelashes, as well as symptoms such as ocular dryness, burning, and foreign body sensation are common in the general population, especially considering the mean age of our cohort, suggesting a possible overlap with ocular surface diseases such as dry eye and meibomian gland dysfunction. However, all patients confirmed a worsening in ocular symptomatology following onset of the disease. In fact, a significant correlation between the presence of blepharitis findings and SPEED questionnaire score was observed (p < 0.0001). We hypothesize that these symptoms may be related to the activity of the SARS-CoV-2 on the ocular surface epithelial cells and glands, causing a microenvironment alteration and consequent instability of the tear film, in particular in patients with pre-existent ocular surface alterations. A similar finding was observed in the oral mucosae, in patients reporting oral symptoms such as dry mouth and amblygeustia, due to damage to the salivary gland caused by the SARS-CoV-2 26 . The SPEED questionnaire used to evaluate the symptomatology of patients was validated in comparison with the most diffuse Ocular Surface Disease Index (OSDI) questionnaire. In fact, the SPEED questionnaire was able to discriminate between asymptomatic and symptomatic subjects related to dry eye disease 27 . However, the mean total score of the SPEED questionnaire in our group was not particularly high, demonstrating mild ocular symptomatology in the evaluated patients.
The Schirmer I test without anesthesia is a standardized test offering an assessment of stimulated reflex. tear flow, particularly recommended to confirm severe aqueous deficiency dry eye disease 28 . In our group, the mean value for the Schirmer I test did not demonstrate a reduction in tear volume. Nevertheless, we were able to collect a sufficient tear volume to perform RT-qPCR analysis.
Previous reports have documented ocular involvement in patients with confirmed COVID-19, in particular unilateral or bilateral follicular kerato-conjunctivitis with moderate hyperemia and conjunctival injection, corneal sub-epithelial infiltrates, mild eyelid edema, clear watery discharge, photophobia, burning eye, and foreign body sensation 8,9 . Furthermore, pseudomembranous and hemorrhagic conjunctivitis was reported in a patient with severe COVID-19 after the beginning of symptoms. No patients have described a loss of vision, and all cases have reported that the conjunctivitis was resolved in few days. In rare cases, ocular manifestations may represent the first clinical presentation of COVID-19 8,9 . In our group, no patients reported ocular conjunctivitis as the first clinical manifestation of COVID-19. In a large Chinese cohort study on 1099 patients, the presence of "conjunctival congestion" was found in 0.8% of the patients 7 . Although kerato-conjunctivitis is not a common manifestation, other studies documented the onset of ocular symptoms and manifestation, such as conjunctival hyperemia, chemosis and epiphora in 27-31% of patients with COVID-19 10,11 . Zhou et al. documented ocular findings in eight patients of a group of 121 with confirmed COVID-19, reporting an interesting correlation between ocular involvement and the severity of COVID-19 12 . In accordance with previous reports, we noted the occurrence of ocular symptoms in 10 patients (34.5%), conjunctival hyperemia and or chemosis in seven patients (24.1%), and blepharitis findings in eleven patients (37.9%); furthermore, conjunctival findings were significantly correlated with severity of the disease (p = 0.002), but not with the duration of the disease, while blepharitis findings were significantly correlated with duration of the disease (p = 0.003). Previous studies observed that conjunctival hyperemia and chemosis most commonly occurred in patients with severe systemic disease 11,12 . Several factors may explain the correlation between conjunctival involvement and severity of COVID-19 such as the viral load, and the conjunctival immune response [23][24][25] ; however its real pathogenesis is still not clear. Moreover, conjunctival involvement may be part of the nonspecific, systemic manifestations of diseases like renal failure, cardiopulmonary failure, and carbon dioxide retention, rather than infectious conjunctivitis of COVID-19 13 . Table 4. Correlation between ocular findings and duration and severity of disease. a Conjunctival hyperemia and chemosis; conjunctival chemosis; b Ocular secretion; lid margin hyperemia and/or telangiectasia; crusted eyelashes, meibomian orifices abnormalities; c Values from the right eye. *Spearman rank correlation analysis; Significant data are reported in bold. www.nature.com/scientificreports/ Wu et al. showed that white blood cell, neutrophil counts, procalcitonin, C-reactive protein and lactate dehydrogenase were higher in patients who presented with conjunctival involvement when compared to those without any ocular findings 11 . On the other hand, it is well documented that ocular surface disease affects up to 60% of patients admitted to the intensive care unit, due to exposure keratopathy, reduced blink reflex and drying effects of high flow oxygen 29 6 . These studies demonstrated that the SARS-CoV-2 RNA could be detected from the normal ocular surface of COVID-19 patients, which suggests that the virus is spread through conjunctival contact, and the kerato-conjunctivitis could occur in a predisposed eye and/or when the viral load on the ocular surface is particularly high 6,12 . In the present study, tear analysis did not document the presence of SARS-CoV-2; however, several factors may clarify the negativity of this test. Firstly, tear collection during the initial stages of infection demonstrates an increased probability of SARS-CoV positivity 30 . Only five of the evaluated patients (17.2%) were in the early stage of the disease. Furthermore, the Schirmer I test strips can collect an appropriate volume of tears, but these may contain few epithelial cells and secretions, reducing the probability of identifying the virus. The main limitations of the present study are the small sample size of patients and the different severities and durations of disease at the ophthalmologic assessment and tear collection. Moreover, we did not compare the SPEED questionnaire results with previous results taken prior to the onset of the disease. In conclusion, our data demonstrate the occurrence of different ocular manifestations in patients with COVID-19 but a low probability of detecting the virus in tears and on the ocular surface, when using Schirmer strips as collecting method, especially in patients with a long duration of the disease. Furthermore, it should be noted that ocular surface signs, other than conjunctivitis, can be present in patients affected by COVID-19.

Methods
This observational study was conducted in 29 hospitalized patients who were admitted to the COVID center at Policlinic Hospital of the University of Messina, Messina, Italy between the 12 th April and 14 th of May 2020. Informed consent was obtained from each patient. The research was carried out in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the University of Messina (protocol number 36/20). All patients tested positive for SARS-CoV-2 according to the WHO interim guidelines 31 . A confirmed case was defined as a positive RT-qPCR result in samples from nasopharyngeal and pharyngeal swabs.
The clinical data of patients, including, age, sex, anamnesis, signs, symptoms, recent exposure history, laboratory findings, and chest computed tomography (CT) scans, were obtained from electronic medical records. The severity of COVID-19 was established according to the recent guidelines 31 .
All patients underwent an ophthalmologic assessment comprising the SPEED questionnaire regarding the frequency and severity of dryness and/or foreign body sensation, ocular pain, burning, and eye fatigue. Each symptom was evaluated as follows: for frequency, from 0 = never to 3 = constant; and for severity, from 0 = absence to 4 = intolerable. The composite score of the questionnaire was obtained by summing up the scores of the frequency and severity parts of the questionnaire; the total score ranged from 0 to 28 32 .
All patients were questioned about previous ocular surgeries, eye disease, use of eye drops, and use of distance spectacles for refractive errors such as myopia, hyperopia, and astigmatism. The anterior segment and the ocular surface of both eyes were examined by an experienced ophthalmologist (AM) using a portable slit lamp (HEINE HSL 150, HEINE Optotechnik, Herrsching, Germany). Conjunctival hyperemia was classified according to the Efron Grading Scales for Contact Lens Complications 33 .
The diagnostic criteria considered for blepharitis comprised the presence of symptoms such as burning, tearing, itching, irritation, photophobia, and findings like hyperemia and/or telangiectasia of eyelid margin, abnormal deposits at the base of the eyelashes, abnormalities of meibomian orifices such as capping, pouting, and obliteration 34 .
Body temperature was measured immediately before the ophthalmologic assessment with a thermo-scan thermometer.
For the Schirmer I test, filter paper strips (Test di Schirmer, Alfa Intes, Casoria, Italy) were applied at the junction between the outer and middle third of the lower lid in both eyes. The moisturized length of the strip was measured after five minutes 35 ; afterwards the strips were stored in vials containing universal viral transport media and delivered to the diagnostic laboratory and tested for the presence of SARS-CoV-2. The nasopharyngeal swab was performed on the same day in all patients. Ribonucleic Acid (RNA) extraction was carried out using the NucliSens EasyMag system (bioMérieux, Marcy l'Etoile, France). From a 200-μL tear sample, an aliquot of 100 μL was used for RT-qPCR (Allplex 2019-n CoV Assay Seegene) to identify a target gene specific for SARS-CoV-2 (E gene, RdRP gene, N gen). Data analysis was performed using CFX96, Bio-Rad. An internal control was employed to verify the quality of the extraction in all samples.