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

BackgroundThe 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 findingsTo 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 & PLUSMN; 11 years, 40% female) and qPCR (n = 5 corneas, 80 & PLUSMN; 12 years, 40% female). Additionally, 5 freshly excised corneas (80 & PLUSMN; 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (10(6) 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 & PLUSMN; 10 years, 29% female) and 26 healthy donors (79 & PLUSMN; 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 & PLUSMN; 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 x 10(8) [95% CI: 6.4 x 10(7) to 2.4 x 10(8)] to 3.0 x 10(9) [1.4 x 10(9) to 5.3 x 10(9)], p = 0.0039 and 2.2 x 10(7) [1.4 x 10(7) to 3.6 x 10(7)] to 5.1 x 10(7) [2.9 x 10(7) to 7.5 x 10(7)], p = 0.0117, respectively) and in corneoscleral rim (4.5 x 10(9) [2.7 x 10(9) to 9.6 x 10(9)] to 3.9 x 10(10) [2.6 x 10(10) to 4.4 x 10(10)], p = 0.0039 and 3.1 x 10(8) [1.2 x 10(8) to 5.3 x 10(8)] to 7.8 x 10(8) [3.9 x 10(8) to 9.9 x 10(8)], 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. |ConclusionsI n this study,weobservedtheexpressionof SARS-CoV-2receptorsandactivatorsat thehumanocularsurfaceanda variableincreasein viralRNAcopies24hoursafterexperimen-talinfectionof freshlyexcisedhumancorneas.WealsofoundviralRNAonlyin a verylimitedpercentageof donorswithpositivenasopharyngealPCR.Thelowrateof positivityin donorsdiagnosedwithCOVID-19callsintoquestiontheutilityof donor selection algorithms

Automated summary

In this study, the capacity of human cornea to be infected by SARS-CoV-2 was assessed, and it was found that corneas can be infected and replicate the virus ex vivo. The risk of corneal contamination by SARS-CoV-2 in donors diagnosed with COVID-19 was also found to be low.