key: cord-0871075-9byus8e6 authors: Sawant, O. B.; Singh, S.; Wright, R. E.; Jones, K. M.; Titus, M. S.; Dennis, E.; Hicks, E.; Majmudar, P. A.; Kumar, A.; Mian, S. I. title: Prevalence of SARS-CoV-2 in human post-mortem ocular tissues date: 2020-10-06 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.10.05.20201574 sha: e7d039afc4e6d551e11feb924bfe2ddd8352e45f doc_id: 871075 cord_uid: 9byus8e6 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. The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 49 -2 (SARS-CoV-2) has been the current focus of research as it has significantly disrupted many 50 livelihoods. The SARS-CoV-2 virus is highly infectious and transmitted primarily through 51 respiratory droplets and upon contact with infected persons. Studies to date have suggested that 52 COVID-19 patients have a high viral load in the upper respiratory tract at disease onset [1, 2] . 53 There is a strong possibility the virus may contaminate the ocular surface via respiratory droplets 54 after coughing, sneezing, and hand-to-eye contact. In January 2020, an ophthalmologist contracted 55 COVID-19 from an asymptomatic glaucoma patient; the initial indication was that the ocular 56 surface served as a mediator of viral infection [3] . Studies have shown that SARS-CoV-2 may 57 cause conjunctivitis, and viral RNA has been detected in tears and retinal biopsies of patients [4] [5] [6] [7] [8] [9] [10] [11] . Conjunctival manifestations have been reported at rates as low as 0.8% [6] in the 59 largest retrospective study from China, and at rates of 6% [9], 32% [5] , and as high as 66% [10] 60 in other studies. These reports indicate that the ocular surface may act as a possible mode of disease 61 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. Ocular surface 64 Although it is believed that SARS-CoV-2 primarily transmits via respiratory droplets, 71 extra-respiratory transmission via blood is theoretically possible because studies have shown the 72 presence of SARS-CoV-2 RNA in blood samples [14, 15] . Hence, there is a strong possibility that (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020 . . https://doi.org/10.1101 personal protective equipment (PPE) and standard disposable instruments were used for eye 94 recovery purposes. A postmortem nasopharyngeal (NP) swab and blood were collected for 95 serological analysis. The left eye was recovered first, followed by the right eye, with disinfection 96 of the instrument taking place between the recovery of each eye. The ocular tissue was 97 swabbed starting with the left eye. The left eye was opened, and the anterior surface was soaked 98 in 5% povidone-iodine (PVP-I) (Aplicare, Meriden, CT) for five minutes. The eye 99 was thoroughly flushed with sterile eyewash solution (Medique Products, Fort Myers, FL). The 100 lid speculum was carefully inserted, followed by a conjunctival swab collection, and the tissue was 101 excised 360°. A sample of the conjunctival tissue was placed in a 5mL microcentrifuge tube 102 (Eppendorf AG, Hamburg, Germany) containing 4% paraformaldehyde (PFA). The remaining 103 tissue was soaked in PVP-I for another five minutes, followed by a sterile saline flush. A swab of 104 the anterior cornea was taken by touching the swab to the cornea's epithelial surface (referred to 105 as anterior cornea). Next, the whole eye was enucleated and positioned in the RE-One chamber 106 (RE-One Sagl, Lugano, Switzerland, acquired via Medical Innovation Partners International, 107 Dallas, TX) for corneal excision. The cornea was removed and thereafter swabbed on the cornea's 108 endothelial surface (referred to as posterior cornea). The cornea was stored in a container with 4% 109 PFA. The iris and lens were carefully removed, and the iris stored without storage media at -80 110 o C. The vitreous was swabbed as well. The RE-One chamber was assembled, and 111 the posterior part was submerged in a fixative solution. The instruments were disinfected 112 using CaviWipes TM (Metrex, Orange, CA). The tissue recovery for each donor resulted in nine 113 swabs for SARS-CoV-2 PCR testing (one NP, two conjunctiva, two anterior corneas, two posterior 114 corneas, and two vitreous) and a postmortem blood sample for measuring anti-SARS-CoV-2 IgM 115 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020 . . https://doi.org/10.1101 and IgG using ELISA. The nine swabs and blood samples were immediately shipped to Eurofins 116 VRL Laboratories (Centennial, CO USA) for SARS-CoV-2 testing by qRT-PCR. To assess the presence of SARS-CoV-2 in ocular tissue, we evaluated tissues that were procured 132 for surgical purposes but later ruled out due to positive post-mortem tests or signs/symptoms of 133 COVID-19. Corneal (n =66) and scleral (n = 66) tissue from 33 different donors were dissociated 134 using the bead lysis method followed by total RNA extraction using Trizol reagent (Invitrogen, 135 Carlsbad, CA) per the manufacturer's instructions. cDNA was prepared using 1mg of RNA using 136 a Maxima first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA) per the 137 manufacturer's instructions. Viral RNA was detected in the cDNA using SARS-CoV-2 138 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint nucleoprotein gene-specific Taqman probe primers purchased from Integrated DNA Technologies 139 (Coralville, IA). The primer sequence used in the assay was adopted from the Chinese Center for washes, 10 minutes each) and the slides were mounted in Vectashield anti-fade mounting medium 160 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. 164 PCR testing on surgical rule-out tissues 165 To detect the presence of SARS-CoV-2 RNA, we isolated RNA from the sclera and cornea Table 1 . The highest positivity rate of 17% for sclera and 11% for cornea 178 was observed in Group 1. In Group 2, 12% of scleral samples and 15% of corneal samples were 179 positive for SARS-CoV-2 viral RNA. None of the tissues from the two donors in Group 3, who 180 had close contact with a COVID-19 patient, showed any detectable level of SARS-CoV-2 RNA. Detecting SARS-CoV-2 in donors that died due to COVID-19 182 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint After observing 13% (17 out of 132) SARS-CoV-2 RNA prevalence rate in surgical rule-out 183 tissues, our next goal was to systematically evaluate the presence of SARS-CoV-2 in different 184 ocular layers of research consented donors that died due to COVID-19. We analyzed ocular swabs 185 from 10 different donors with a median age of 66 (range 46-90). Donor demographics and pre-186 mortem testing results are provided in Table 2 . Detailed case summaries with a timeline of disease 187 progression and testing results are provided below. The testing results are also summarized in 188 Table 3 . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . Surprisingly, the post-mortem NP swab test was negative for SARS-CoV-2 RNA. However, to our surprise, all ocular tissue swabs were negative. Case #6: A Hispanic male in his late 60s was admitted to the hospital after being found 226 unresponsive. The patient was deemed positive for COVID-19 via PCR assay on the next day after 227 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. Three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested 267 positive for SARS-CoV-2 RNA, exhibiting a positivity rate of 15% for conjunctiva, 5% for 268 anterior corneal surface, 25% for posterior corneal surface, and 15% for vitreous (Table 3) . Right corneal tissue that were procured without any PVP-I disinfection and showed overall 271 stronger positivity (lower CT value) for SARS-CoV-2 was immunostained to detect viral antigens. Our IHC data showed that coinciding with positive viral RNA, SARS CoV-2 Envelope protein 273 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint (Fig. 2) , and Spike protein (Fig. 3) were detected in the COVID-19 patient's right corneas (Cases 274 7-9). The positive staining was primarily observed in epithelial layer of the cornea. In contrast, no 275 positivity for spike protein was visualized in COVID-19 negative patient corneas (healthy control). Non-specific antibody binding was ruled out by staining sections with a secondary antibody only 277 as control. Also, the specificity of antibodies was validated by infecting cells with live SARS- CoV-2 and staining them for spike protein (data not shown). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint explore the possibility of transmission via ocular tissues and concentration dependent anti-virulent 296 activity of PVP-I. In the context of the route of administration, it has been proposed that the virus travels via 298 direct airborne transmission since the entire ocular surface including the anterior cornea, tear film 299 and conjunctiva communicates with air, similar to the nasal mucosa. Another proposed path of 300 infiltration is the nasolacrimal route, facilitating the transport of viral particles from the respiratory 301 tract to the ocular surface or vice versa [17] . These potential routes of infiltration may justify 302 reports suggesting that SARS-CoV-2 causes conjunctivitis in 9, 303 10, 18] . Incidences of conjunctivitis in these reports are mainly in the range of 0.8-6% but they 304 can be as high as 32% and 66% (for a detailed review, please refer to [19] ). Although the primary route of SARS-CoV-2 infiltration could be via respiratory droplets, 306 a potential non-respiratory route of transmission cannot be ignored. To our surprise, we observed 307 a higher prevalence of SARS-CoV-2 RNA in the posterior corneal (endothelial) surface than from 308 any other ocular surface swabs (Table 3) . This finding and current literature highlighting the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint remain intriguing, and further studies are required to decipher the detailed mechanism. As 319 postulated by Ang and colleagues [20] , a contributing factor for this finding could be higher 320 antimicrobial activity on the ocular surface. Tear film contains antimicrobial protein lactoferrin, 321 which has been postulated to prevent bindings of SARS-CoV-2 to the ACE2 receptors on anterior 322 corneal and conjunctival surfaces. An initial study goal was to detect the effectiveness of PVP-I in inactivating the SARS-324 CoV-2. Therefore, we recovered right eyes without PVP-I exposure, and left eyes were recovered 325 following EBAA-recommended double PVP-I soak procedure. We are unable to make conclusive 326 remarks about the effectiveness of PVP-I, due to the small sample size (10 cases) of this study. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020 . . https://doi.org/10.1101 corneal epithelial cells. We previously showed that corneal epithelia cells elicit innate responses 342 upon viral stimuli such as ZIKV [28] or PolyI:C, a viral mimic of dsRNA [29] . However, further 343 studies are necessary to understand if potential transmission of SARS-CoV-2 through the ocular 344 surface is possible. In summary, we report the presence of SARS-CoV-2 RNA and proteins in the ocular Network for referring COVID-19 donors for this study and Eversight's Donation Support Center 364 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint *Ct refers to number of PCR cycles All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 6, 2020. . https://doi.org/10.1101/2020.10.05.20201574 doi: medRxiv preprint Clinical and virological data of the first cases of COVID-19 in Europe: a case 371 series SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Can the Coronavirus Disease 2019 (COVID-19) Affect the Eyes? 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The 423 Lancet. Infectious diseases Infection and Replication of Influenza Virus at the Ocular Surface Interferon-stimulated gene 15 (ISG15) restricts Zika virus replication in primary 427 human corneal epithelial cells. The Ocular Surface Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in 429 human corneal epithelial cells No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity