Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study

Abstract

Background The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. Methods and findings To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. Conclusions In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. Trial registration Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/. Corantin Maurin and colleagues investigate the capacity of human cornea to be infected by SARS-CoV-2 virus and evidence for viral detection in corneas from donors with a positive test for SARS-CoV-2 infection. Author summary Why was this study done? Corneal transplantation is by far the most common transplantation procedure in the world, and there is a severe lack of corneal donation to respond to the increasing patient need. The persistent Coronavirus Disease 2019 (COVID-19) pandemic has dramatically worsened this situation by limiting the access to corneas during a long period of the pandemic and finally increasing the long list of contraindications to corneal donation based solely on an exacerbated precautionary principle. Exploration of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) risk of transmission through corneal transplantation is mandatory to adapt the donor selection strategy and improve corneal tissue procurement in the persisting COVID-19 crisis. What did the researchers do and find? We explored and observed the expression of 4 proteins related to molecular pathways allowing SARS-CoV-2 cellular entry at the ocular surface (cornea, bulbar conjunctiva, 10 samples). These proteins were differentially located in the studied tissues, and their expression was variable among donors. The experimental infection of 5 freshly excised corneas with highly concentrated SARS-CoV-2 viral solution promoted only moderate and variable viral RNA multiplication in corneal samples. Twenty-four hours after inoculation, a significant increase in total and positive strand viral RNA was observed in the epithelium of central cornea (p-value: 0.0039 for and 0.0117, respectively) and in corneoscleral rim (p-value: 0.0039 and 0.0391, respectively). We explored the presence of SARS-CoV-2 viral genetic material in corneas and respective storage media from donors tested positive or negative to COVID-19 PCR on nasopharyngeal swab. All samples from the 159 donors not contaminated by COVID-19 were negative for SARS-CoV-2 RNA. SARS-CoV-2 RNA was detected in only 3 corneas out of 28 corneas (11%) from donors diagnosed with COVID-19. What do these findings mean? Our results support that the ocular surface expresses in a variable way the proteins involved in SARS-CoV-2 cellular entry and infection. We observed a moderate and variable increase in total and positive viral RNA after experimental infection of human freshly excised corneas. A very low rate of positivity in donors diagnosed with COVID-19 was observed, supporting a low risk of SARS-CoV-2 presence in donor corneas. This work suggests that it would be beneficial to procure corneas from donors tested positive to COVID-19 (nasopharyngeal swabs), test their corneal storage media, and transplant the negative corneas.