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/.

IgA-Based Secretory Response in Tears of COVID-19 Patients: A Potential Biomarker of Pro-Inflammatory State in Course of SARS-CoV-2 Infection

“…Secretory IgA antibodies are present in human mucosal secretions such as tears, milk and saliva to provide protection against infection through IgA-dependent local protective factors [ 2 ]. However, relatively little is known about immunity to SARS-CoV-2 at the ocular surface, even though this may be an important entry portal [ 3 , 4 , 5 , 6 ]. Thus, there is a need for laboratory serological assays that can measure local antibody responses at the ocular surface for the identification of seroconversion in COVID-19 patients.…”

Investigation of SARS-CoV-2 in Postmortem Ocular Tissues and Evaluation of Its Effects on Corneal Donation

“…All had nasopharyngeal swab samples tested positive for the RT-PCR test for SARS-CoV-2. Maurin et al 32 found SARS-CoV-2 RNA in only 3 of 28 corneas (11%) received from donors, who were diagnosed with COVID-19. In a study by Aiello et al, 31 none of the 14 patients had corneal epithelial samples The results were considered statistically significant in cases where the P value was ,0.05.…”

SARS‐CoV‐2 cellular tropism and direct multiorgan failure in COVID‐19 patients: Bioinformatic predictions, experimental observations, and open questions

“…Singh et al, 2022). Additionally, SARS-CoV-2 was able to infect human cornea cells ex vivo (Maurin et al, 2022). Productive viral replication in postmortem samples and eye organoid cultures was reported, and, importantly, limbal cells were found to be more permissive than corneal cells to SARS-CoV-2 infection (Eriksen et al, 2021).…”