key: cord-0943998-du4ghqqf
authors: Sawant, Onkar B.; Singh, Sneha; Wright, Robert Emery; Jones, Kayla M.; Titus, Michael S.; Dennis, Eugene; Hicks, Eric; Majmudar, Parag A.; Kumar, Ashok; Mian, Shahzad I.
title: Prevalence of SARS-CoV-2 in human post-mortem ocular tissues
date: 2020-11-08
journal: Ocul Surf
DOI: 10.1016/j.jtos.2020.11.002
sha: 531e929837ba9c1257809ca8e47e759bc1bbd23b
doc_id: 943998
cord_uid: du4ghqqf

BACKGROUND: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. METHODS: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors. FINDINGS: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ∼13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP–I) disinfection. INTERPRETATIONS: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular 28 tissues. Coronavirus -2 (SARS-CoV-2) has been the current focus of research as it has significantly 50 disrupted many livelihoods. The SARS-CoV-2 virus is highly infectious and transmitted 51 primarily through respiratory droplets and upon contact with infected persons. Studies to date 52 have suggested that COVID-19 patients have a high viral load in the upper respiratory tract at 53 disease onset [1, 2] . There is a strong possibility the virus may contaminate the ocular surface via 54 respiratory droplets after coughing, sneezing, and hand-to-eye contact. In January 2020, an 55 ophthalmologist contracted COVID-19 from an asymptomatic glaucoma patient; the initial 56 indication was that the ocular surface served as a mediator of viral infection [3] . Studies have 57 shown that SARS-CoV-2 may cause conjunctivitis, and viral RNA has been detected in tears and 58 retinal biopsies of COVID-19 patients [4] [5] [6] [7] [8] [9] [10] [11] . Conjunctival manifestations have been reported at 59 rates as low as 0.8% [6] in the largest retrospective study from China, and at rates of 6% [9], 60 32% [5] , and as high as 66% [10] in other studies. These reports indicate that the ocular surface 61 may act as a possible mode of disease transmission.

SARS-CoV-2 relies on angiotensin-converting enzyme-2 (ACE-2) as its receptor on 63 human cells, along with TMPRSS2 or Furin protease for viral entry into host cells [12] . Ocular 64 surface cells of the cornea and conjunctiva epithelial cells have been shown to express ACE-2 65 and TMPRSS2 [13, 14] . Hence, based on the recent evidence, potential SARS-CoV-2 66 transmission through the ocular surface remains a significant concern. The route of transmission 67 and infiltration of the virus within the ocular tissue is still unknown. Answering these questions 68 are of critical importance concern to ophthalmologists, eye banking industry, and the field of were used for eye recovery purposes. A postmortem nasopharyngeal (NP) swab and blood were 95 collected for serological analysis. The left eye was recovered first, followed by the right eye, 96 with disinfection of the instrument taking place between the recoveries of each eye. The ocular 97 tissue was swabbed starting with the left eye. The left eye was opened, and the anterior surface 

To assess the presence of SARS-CoV-2 in ocular tissue, we evaluated tissues that were procured 134 for surgical purposes but later ruled out due to positive post-mortem tests or signs/symptoms of 135 COVID-19. Corneal (n =66) and scleral (n = 66) tissue from 33 different donors were dissociated 136 using the bead lysis method followed by total RNA extraction using Trizol reagent (Invitrogen, 137 Carlsbad, CA) per the manufacturer's instructions. cDNA was prepared using 1mg of RNA using Ct values >37 were considered negative. The whole-genome SARS-CoV-2 RNA was used as a 150 positive control, while a no-template mixture was used as a negative control for the assay. Table 1 . Table 3 . for anterior corneal surface, 25% for posterior corneal surface, and 15% for vitreous (Table 3) . 

Although the primary route of SARS-CoV-2 infiltration could be via respiratory droplets, 317 a potential non-respiratory route of transmission cannot be ignored. To our surprise, we observed 318 a higher prevalence of SARS-CoV-2 RNA in the posterior corneal (endothelial) surface than 319 from any other ocular surface swabs (Table 3) . This finding and current literature highlighting 

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