Does SARS-CoV-2 replicate in ocular surface epithelia?
Corroborating laboratory evidence thought to show that SARS-CoV-2 infects ocular surface epithelia has now been published by many groups, using a combination of approaches including: RT-PCR analyses on ocular samples (eg, tears, swabs and cadaveric eyes); determining the ocular surface tropisms of SARS-CoV-2 by analysing the expression of cell surface receptors, including angiotensin converting enzyme-2 (ACE2) and type 2 transmembrane serine protease (TMPRSS2), among other secondary proteins; and in models of infection within human cell lines and animals. However, the evidence to support SARS-CoV-2 infection of ocular surface epithelia is far thinner than generally appreciated, with interpretations of results impacted by neglecting the limits of biological plausibility, absence of control data, and doubtful transportability assumptions between the wet bench and the dynamics of infection in real-world settings. Such limitations become clearer by specifying the model of live human infection that would most accurately describe active SARS-CoV-2 replication within ocular surface epithelia: (a) exposure of ocular surface epithelia to SARS-CoV-2, either by aerosols and/or by direct contact; (b) evasion of robust ocular surface protections, including mechanical (eg, tear washout) and chemical (eg, mucosal immunoglobulins, complement and antimicrobial peptides)87 defences; and (c) attachment to, invasion of, and replication within ocular surface epithelia, resulting in pathognomonic cytopathic effect.
PCR analysis of ocular surface samples
Possible ocular surface tropisms of SARS-CoV-2 have been studied most commonly via RT-PCR analyses of ocular samples, including conjunctival swabbing and tears. The proportion of patients with COVID-19 who have returned positive RT-PCR tests from the ocular surface has ranged from 0% to 57%,16 88–98 with the large range of values attributable once again to differences in sampling fractions, highly variable case definitions for both COVID-19 and ‘conjunctivitis’, and test collection methods employed (including live vs postmortem testing).99 Furthermore, results may also be contingent on the testing laboratory, since clinical laboratories are often subject to different regulatory requirements regarding validation and limits of detection than when compared with research laboratories.100 Correctly interpreting RT-PCR tests also requires nuance and understanding of the methodology to avoid misinterpretation. RT-PCR of any surface only tests for the presence of the nucleic acid, and does not necessarily indicate infectious virus. That is, RT-PCR cannot confirm whether the viral RNA discovered represents intact virus capable of replication, for example, in the preocular tear film, or whether there is actual viral replication in ocular surface epithelium. SARS-CoV-2 RNA has been identified by RT-PCR on windowsills, air vents, bedrails and shoes,101 102 and no one would suggest the virus is replicating on these acellular, nonliving surfaces. Therefore, detecting SARS-CoV-2 RNA on the eye surface by RT-PCR may be no more significant than finding the RNA on the same person’s shoes. Rather, positive tests merely provide an indication that viral RNA has been recovered from the sampled area, and neither its source nor viability can be determined with certainty. Likewise, negative tests should not be construed as definitive absence of virus, since false-negatives may arise due to poor collection technique, the clinical window in which sampling occurred, and the potential need for repeated collection.100 103
Clinical correlations between conjunctivitis and a positive conjunctival RT-PCR test remain poor at best. For example, Azzolini et al conducted a cross-sectional study on 91 hospitalised patients with COVID-19 (confirmed on nasopharyngeal RT-PCR), reporting that among 52 (57%) patients whose conjunctiva tested positive, only 3 (6%) had conjunctival hyperemia and 3 (6%) had ocular ‘secretions’.104 The authors quite correctly suggested that viral RNA detected from the ocular surface could have been sourced elsewhere, for instance from the aerosolised microenvironment around the face (particularly patients on mechanical ventilation), or from the lacrimal glands or ocular surface vasculature in the setting of systemic infection and viremia. To date, only one report has provided evidence of infectious virus—not just RNA—directly isolated from the conjunctiva of a patient with COVID-19. Colavita et al reported the case of a 65-year-old patient with laboratory-confirmed COVID-19, who presented with fever, mild upper respiratory symptoms, and bilateral conjunctival hyperemia and chemosis.105 The authors took a conjunctival swab on day 3 of hospitalisation, and inoculated its contents within Vero E6 kidney epithelial cells, observing viral cytopathic effects 5 days later. Concurrently, viral RNA was isolated from spent cell media using RT-PCR, along with positive RT-PCR tests on ocular swabs collected throughout hospitalisation. While this report provides a compelling account of in vitro SARS-CoV-2 infectivity, the authors did not perform PCR for other viruses. Furthermore, the cytopathic effect observed in cell culture after inoculation by the clinical sample could not be definitively attributed to SARS-CoV-2, as immunodetection assays, including those testing for antigens of other viruses, were not performed.
Ocular surface tropisms of SARS-CoV-2
It is well-established that the two canonical transmembrane receptors for SARS-CoV-2 infection in human epithelial cells are ACE-2 and TMPRSS2.106–112 ACE-2 serves as a direct viral binding site, while TMPRSS2 is involved in cleaving the SARS-CoV-2 spike (S) protein at S1/S2 and S2, thereby priming the virus for cellular entry. Whether the ocular surface epithelium expresses co-localised ACE-2 and TMPRSS2 in the requisite quantities to permit routine SARS-CoV-2 infection remains controversial, with divergent perspectives captured by studies reporting both high111 113–119 to essentially negligible120 121 expression of these proteins. Furthermore, it is not known whether the expression of these receptors may vary with various health states (eg, systemic COVID-19 infection vs non-infection), with pre-existing eye disease, or in the setting of comorbidities where ACE-2 plays a pathophysiological role (eg, cardiovascular disease). There is some limited evidence to suggest that ACE-2 and TMPRSS2 may be localised to ocular surface tissues. In one such study where immunohistochemistry was performed on postmortem surgical specimens from healthy individuals, Zhou et al demonstrated diffuse presence of ACE-2 and TMPRSS2 in corneal and conjunctival epithelia.113 Curiously, however, ACE-2 staining was far more prominent within the basal corneal epithelium relative to the outermost apical layers, with this staining asymmetry most pronounced at the corneal limbus. Furthermore, while the authors showed putative ACE-2 expression within conjunctival crypts, it was not clear whether the isotype control captured the same conjunctival crypts shown with the primary antibody.
Data on whether SARS-CoV-2 can infect ocular surface epithelium also remain sparse, even under ideal experimental conditions. In one immunohistochemistry study of ex vivo human conjunctival explant cultures by Hui et al, a clinical SARS-CoV-2 isolate was used to inoculate three human conjunctival explant cultures, with an exponential rise in viral titers 48 hours post infection (hpi) strongly suggestive of infection.122 However, when anti-SARS-CoV-2 nucleoprotein was used to stain the conjunctival specimens at 48 hpi, viral antigen appeared only within the conjunctival substantia propria, and not in the epithelium. This may be because explant cultures lack a confluent epithelial barrier—virus in the culture media bathes and can access cells within the tissue from any side, including the stromal side of the explant. In another ex vivo human explant study, Miner et al showed that a clinical isolate of SARS-CoV-2 did not replicate within human corneal donor tissue recovered from seven non-COVID-19 infected patients, confirmed using serial RT-PCR and plaque assays, and as compared with a positive control using HSV-1 to infect the same specimens.123 Furthermore, the authors reported that SARS-CoV-2 did not replicate in the residual conjunctival and limbal tissue that remained attached to the explanted corneas. Unsurprisingly, there is similarly conflicting evidence as to whether SARS-CoV-2 can be detected from human donor corneas retrieved from patients known to be COVID-19 positive at the time of death. One immunohistochemical study by Sawant et al reported SARS-CoV-2 spike and envelope protein in the epithelium of three donor corneas that had not undergone disinfection with povidone-iodine.124 Meanwhile, RT-PCR analyses from independent groups did not identify SARS-CoV-2 RNA from iodine-disinfected donor corneas.125 126
The ocular surface as a passive conduit to nasopharyngeal SARS-CoV-2 infection
Consideration of the ocular surface as both contagion and anatomical conduit has naturally invited speculation that nasopharyngeal SARS-CoV-2 infection may occur through the nasolacrimal system.127 128 This theory came to prominence early during the pandemic, when Dr Wang Guangfa, a distinguished SARS expert, developed COVID-19 after visiting a Wuhan hospital in January 2020.129 Having worn a personal protective gown and an N95 mask, Dr Wang attributed his infection to a lack of protective eyewear, recalling bilateral conjunctival congestion prior to the onset of respiratory symptoms. While other respiratory viruses such as human and avian influenza can cause systemic illness130–132 following conjunctival inoculation, whether the same can be concluded for SARS-CoV-2 remains unclear. Deng et al applied a relatively large inoculum (106 TCID50/mL) of SARS-CoV-2 to the conjunctival surfaces of two rhesus macaques, and reported that both animals developed mild COVID-19 respiratory symptoms, with a continuously detectable viral load sourced from nasopharyngeal and oropharyngeal swabs up to 7 days post inoculation.127 However, virus could not be detected from conjunctival swabs after 1 day post inoculation. Histology was not performed on the conjunctiva at euthanasia, but the repeatedly negative RT-PCR results from conjunctival swabs after 24 hpi suggests ocular surface infection did not occur.
In sum, clarity around ocular involvement in COVID-19 is lacking. Experimental studies have either not been confirmed or would be difficult to reproduce, and existing data do not lend the sort of overwhelming support for ocular surface infection that has been suggested by some retrospective clinical studies. Rather, the weight of current evidence supports a limited role for the ocular surface in viral shedding and transmission. Questions remain regarding how routinely SARS-CoV-2 (and its variants133) infects ocular surface epithelia as compared with other vulnerable cell types (eg, nasopharyngeal epithelia), whether the conjunctival and corneal epithelium may have different susceptibility profiles (eg, due to discordant expressions of cell surface proteins required for viral entry) and the potential for viral carriage and infectivity within the eye among recovered patients.134 135 Furthermore, ex vivo models may not fully capture the real-world viral kinetics on the ocular surface, including the protective effects of the tear film and adnexa.136