Researchers refine methods for detection of SARS-CoV-2 in tears, ocular tissues

Kenneth F. Maxcy, MD, authority on infectious disease, wrote in a study published in JAMA in 1919, referring to the Spanish flu that spread worldwide during 1918-1919:

“Recently the eye has received little or no attention as a factor in the transmission of acute respiratory infections. It has been disregarded in planning measures for the prevention of the spread of contagious diseases. This was especially true in the recent epidemic of influenza.”

In a study, he demonstrated ly that the eye is potentially “a portal of entry of importance in the transmission of acute respiratory infections” and that “an organism introduced into the conjunctival sac may be recovered from the nose in five minutes, from the throat in fifteen minutes, and from the stool in twenty-four hours.” He recommended the use of goggles in addition to face masks because “droplets may not enter the nose or mouth when a face mask is worn, but the conjunctiva remains openly exposed.”

More than 1 year after the COVID-19 pandemic began, many studies have been evaluating the presence of SARS-CoV-2 in the ocular surface and tear secretions, with inconsistent and often contrasting outcomes, mostly due to heterogeneous methodologies. Yet, clarifying the role of the ocular transmission route is important for better risk assessment and prevention of COVID-19 infection through the eye, as well as for testing the ocular tissues intended for transplantation, according to Ashok Kumar, PhD, associate professor in the department of ophthalmology, visual and anatomical sciences at Wayne State University, Detroit.

“The ACE2 receptor, the gateway of SARS-CoV-2 into the cells, is widely distributed in the organs and tissues of the human body, including the ocular tissues. COVID-19 is an assault to all organs, so there is no reason to believe that it does not enter the eye. There is little we know at the moment, and we must continue to systematically collect evidence, do more testing and investigate hypotheses,” he said.

The COVID-19 environment has posed methodological challenges to researchers, often leading to questionable outcomes and underestimation of the presence of the virus in the conjunctiva.

“In many cases, the conjunctival swabs are performed late in the course of the disease. Since the virus load goes up and quickly drops, if you do a conjunctival swab late, it will be negative because the virus has gone,” Kumar said.

Rigorous timing, reproducible methods

In a study conducted by the group of Claudio Azzolini, MD, professor of ophthalmology at University of Insubria in Varese and Como, Italy, SARS-CoV-2 was found on the ocular surface of 52 out of 91 patients (57.1%) hospitalized with COVID-19.

“We found a high quantity of viral material in the majority of patients, something that surprised us since in several previous studies only a very small minority of patients tested positive from conjunctival swabs. The higher rate reported in our study was likely due to the rigorous methods we adopted in sampling and processing the fluid samples,” he said.

He said that all samples were collected by the same ophthalmologist, Elias Premi, MD, at the time still a resident, under the supervision of Simone Donati, MD. Wearing a full-body protective suit, Premi performed a conjunctival swab at the bedside of patients hospitalized for COVID-19 in three ICUs. He also examined the eyelids, conjunctiva and cornea, and scribbled down data on hospitalization timing, serological examinations and type of respiratory device on his gloves, with the information later transferred to a database on a dedicated form on the Eumeda e-health platform.

The sampling procedure was performed by leaning the swab on the caruncula for 5 seconds and then moving it slowly for a further 5 seconds all the way along the inferior fornix. Special DNA- and RNA-free swabs, optimized for virus samples, were used (FLOQSwabs, Copan Diagnostics). All samples were processed by Andreina Bay, MD, in the same laboratory where thousands of body fluid samples from patients with COVID-19 are processed every day, and the time between sample collection and processing was minimized. The same storage medium and the same RT-PCR procedure were used for all samples.

“Looking at the literature, we found that different sampling, laboratories and processing methods were used also within the same study, and in many cases the samples were stored in the refrigerator for 3 days or more. We found that the interval between collection and analysis should be no more than 2 days because viral particles may no longer be detectable at longer intervals,” Azzolini said.

Variabilities in swab testing

The average viral load was widely variable among patients, ranging between 29 copies/µL and 45,000 copies/µL, and slightly variable between eyes when the virus was present in both eyes.

“We found the virus in both eyes of 31 patients, and 22 of them had a slightly different viral load between the two eyes. The cases with the higher viral load were all bilateral, and unilateral cases had a lower viral load. Maybe in unilateral cases the second eye has yet to be infected, or the viral load is so low that it is not detectable,” Azzolini said.

Interestingly, some of the patients with positive conjunctival swab results had negative nasopharyngeal swab results. This was a meaningful finding, indicating that a negative nasopharyngeal swab does not rule out the presence of the virus in other body fluids.

Several studies have shown that false-negative test results may depend on the time interval occurring between symptom onset and the test, but also on the way the test is performed, according to Azzolini.

“Nasopharyngeal swabs must be performed properly, along the appropriate trajectory, reaching the correct location, rubbing and rolling until they are saturated with mucus. They are inherently uncomfortable. The quick touch and go I see sometimes when patients are swabbed in the car, I do not think that is a reliable way of testing for COVID,” he said.

The study was performed in Lombardy, the Italian region with the highest number of COVID-19 cases, and a region that is highly populated, highly industrialized and highly polluted.

“Two weeks before the flare-up of COVID-19 cases in March last year, our regional air quality monitoring stations had reported very high levels of particulate matter. Viruses piggyback on fine particles, enter the respiratory system and also adhere to the conjunctiva. Lombardy is mostly a flat, windless region, where polluted microparticles tend to remain in the air for long and in a high concentration,” Azzolini said.

Testing immune response

While many studies have investigated the presence of viral particles, only a few have investigated the immune response against the virus in the eyes of patients with COVID-19.

Peter Raus, MD, PhD, visiting professor at Vrije Universiteit Brussel, Belgium, analyzed with his team of researchers the presence of SARS-CoV-2 RNA in tears and found a high 26.9% rate of infected samples in 26 patients with COVID-19. He is currently conducting research on SARS-CoV-2-specific immunoglobulin A (IgA) antibodies in tears. In 30 patients with COVID-19, a perfect correspondence was found between IgA antibodies in blood and tears.

“We have the impression that IgA is very important in COVID. It is our first defender against the virus, and if you have a high IgA concentration in tears, throat and lungs, the virus does not get the chance to penetrate and multiply,” he said.

This might explain why COVID-19 tends to be mild or asymptomatic in healthy children, who have higher serum levels of IgA than adults.

“IgA concentration diminishes with age. Children also get lots of common colds, and perhaps they have cross-immunity against coronaviruses, a faster and higher reaction with IgA concentration,” Raus said.

IgA concentration might also explain why a single virus can cause such different outcomes in adults and may help identify the so-called super-spreaders, he said.

“These are just conjectures, but people with low IgA could be those with a higher viral load because they have a poorer defense against the virus. They could be the same people who have virus particles in tears as well as a positive nasopharyngeal swab,” Raus said.

Further research will be conducted with the support of Sanofi to clarify the presence and role of antibodies in tears. It will also include measuring IgA in one eye and PCR in the other eye in a group of patients with COVID-19 to see if there is a correlation between low IgA, high viral load and positive tears.

“If it is confirmed that tears are a good indicator of the concentration of IgA, then we may embark in a spinoff project, developing a test similar to the one we use for adenovirus, a rapid IgA test that people can do by themselves to detect antibodies in tears after vaccination, for instance, to see when they develop immunity and how long it lasts. As far as we know now, the IgA in tears is as sensitive as in serum, and in a few cases, we found IgA in tears even after it had disappeared from serum,” Raus said.

Detecting SARS-CoV-2 in the cornea

A small but noteworthy presence of SARS-CoV-2 RNA, envelope protein and spike protein in the ocular tissues of donors with confirmed COVID-19 infection was found in a multi-institutional study recently published in The Ocular Surface.

“There is a strong possibility that the different ocular tissues exhibit different degrees of SARS-CoV-2 transmission risk. Based on our findings, we strongly recommend the implementation of specific screening protocols to minimize the risk of transplanting eye tissues containing SARS-CoV-2 particles,” Kumar said.

The study included corneal and scleral tissues from 33 donors not eligible for surgery due to positive postmortem tests, premortem signs/symptoms of COVID-19 or close contact with COVID-19 patients. Additionally, 20 eyes of 10 donors who died of COVID-19 and had families that consented to eye donation for research purposes were included. Postmortem nasopharyngeal swab and blood were collected for serological analysis.

A 13% prevalence of SARS-CoV-2 RNA was found in surgical rule-out tissues. Positivity rate varied between 11% and 17% in the sclera and cornea of patients with positive RT-PCR tests and/or signs and symptoms of COVID-19, while no virus particles were found in the tissues of donors who had been in close contact with patients with COVID-19. In the 20 eyes of patients who died of COVID-19, swabs for RT-PCR detection of SARS-CoV-2 were performed on the conjunctiva, anterior and posterior cornea, vitreous and iris.

“To our surprise, we found the highest prevalence of SARS-CoV-2 particles in the posterior corneal surface swabs. This led us to hypothesize a non-respiratory route of transmission, such as blood. The vascular endothelium-enriched ocular tissues could be the source of posterior corneal infectivity. We did not find virus particles in the iris samples but did in some vitreous samples, and we cannot rule out the possibility that the virus might infiltrate through the retinal vasculature,” Kumar said.

He said that the lesser prevalence of SARS-CoV-2 RNA in the anterior cornea might be due to the higher antimicrobial activity on the ocular surface.

“Lactoferrin, which is contained in the tear film, is an antimicrobial protein and might prevent bindings of SARS-CoV-2 to the ACE2 receptors on the anterior corneal and conjunctival surfaces,” Kumar said.

The study also highlighted the potential role of povidone-iodine to inactivate SARS-CoV-2 from donor tissues. The authors noted that the swabs obtained from eyes in which povidone-iodine disinfection was performed as part of the procurement protocol were negative for SARS-CoV-2, and they concluded that this possibility should be further investigated in a larger sample size.

The same group of researchers is now hoping to obtain funding for further studies.

“We are now culturing corneal cells from the corneas of healthy and diabetic subjects. We infect them with SARS-CoV-2 and study whether corneas of people with diabetes are more easily and more severely infected. Because of our published and ongoing studies, my laboratory has become a sort of biorepository of ocular tissues from COVID-19 patients. In collaboration with Eversight, we are receiving increasing number of samples from various eye banks within the U.S. This would allow us to continuously monitor the prevalence of SARS-CoV-2, including the emerging variants in ocular tissues. In addition, we are also studying in animal models how the body gets infected through the eye,” Kumar said.

Effects of vaccines on transplanted corneas

As a corneal specialist, OSN Cornea/External Disease Board Member Preeya K. Gupta, MD, associate professor of ophthalmology at Duke University Eye Center, trusts eye bank screening protocols to ensure provision of safe tissues in this time of COVID-19.

“There are papers showing that SARS-CoV-2 RNA can be found in the corneal and scleral tissue of donors, but as far as we know, testing positive for the virus does not mean that there is active virus in the tissue. It just means that the virus has been there, and I don’t think this poses a significant risk of transmission,” she said.

To her knowledge, no case of infection through corneal transplantation has been reported, but the situation is so new that nothing can be excluded a priori.

“There is still a lot to learn about transmissibility,” she said. “What we have seen to date is that if you use a sterile technique and appropriate precautions, that will reduce transmission.”

Tissue storage in Optisol (Bausch + Lomb) after harvesting should also affect the survival and infectivity of the virus, although this has yet to be determined, she said.

Another concern is that the immune response triggered by the COVID-19 vaccine may cause graft rejection, and cases of robust immune responses in corneal-transplanted patients have been anecdotally reported.

“It happened with two of my corneal graft patients. In the first week after vaccination, they had symptoms of inflammation in their eyes. They came to the office, and in both cases, we treated them successfully with corticosteroids that resolved the inflammatory episode,” Gupta said.

Immune responses after vaccination in patients with corneal grafts are not new and have been seen previously with the vaccines for influenza.

“This is not a reason to avoid vaccination, but as clinicians, it makes sense for us to give patients some guidance. I tell my patients that they should get the COVID vaccine but should closely monitor their eyes for any light sensitivity, redness or pain and contact us right away if they experience any of these symptoms. There is a small percentage of patients who have a more robust immune response that could actually affect their corneal transplant,” Gupta said.

Protecting the eyes

The eyes offer a large area for droplet deposition and virus replication and are connected to the nose through the lacrimal duct, and from there the virus travels easily to the lungs, Kumar said.

“We should not underestimate the eyes as a route for infection, and we should not underestimate the damage the virus might potentially cause by infiltrating the ocular structures. We do not know what may happen in the long term,” he said.

A study by Zeng and colleagues raised the possibility that wearing eye protection could further contain the spread of infection in the general population.

“They found that among patients hospitalized with COVID-19 in Suizhou, China, there was a higher proportion of patients who did not wear glasses than in the general population, suggesting that those who don’t wear glasses may be more exposed to infection. Spectacles seem to protect the eye, and wearing them in crowded environments, such as buses, subways and classrooms, may be advisable,” Azzolini said.

Eye protective strategies have been implemented for frontline workers but are not so easy for ophthalmologists to adopt, particularly with optical instruments.

“We are careful and take all possible protective measures, but goggles, face shields and screens get in the way when using examination instruments,” Azzolini said.

“There have been victims among us. The first doctor to die, Li Wenliang, was an ophthalmologist, and we have had cases in Belgium among ophthalmologists; one of them had his arms temporarily paralyzed,” Raus said.

Most health care workers and an increasing number of patients are now vaccinated, and this makes clinical practice and surgery significantly more relaxed.

“The risk of transmission to a vaccinated person is very low, and we feel a lot more comfortable. People who are not vaccinated are tested prior to surgery, and we are not operating on COVID-positive patients unless it is an absolute emergency,” Gupta said. “Patients also feel much less concerned when they come to see us and have confidently gone back to seeking routine care. Of course, we still wash hands, wear a mask and keep distances as much as possible, but vaccines have brought a huge change in the way we feel.”

• References:
• Armstrong L, et al. Stem Cells Transl Med. 2021;doi:10.1002/sctm.20-0543.
• Azzolini C, et al. JAMA Ophthalmol. 2021;doi:10.1001/jamaophthalmol.2020.5464.
• Caselli E, et al. Biology (Basel). 2020;doi:10.3390/biology9110374.
• Cheong KX. Curr Ophthalmol Rep. 2020;doi:10.1007/s40135-020-00257-7.
• Coroneo MT, et al. Lancet Microbe. 2021;doi:10.1016/S2666-5247(21)00040-9.
• Coroneo MT. Ocul Surf. 2021;doi:10.1016/j.jtos.2020.05.011.
• Higgins TS, et al. JAMA Otolaryngol Head Neck Surg. 2020;doi:10.1001/jamaoto.2020.2946.
• Latiff AH, et al. Ann Clin Biochem. 2007;doi:10.1258/000456307780117993.
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• Maxcy KF. JAMA. 1919;doi:10.1001/jama.1919.02610090020005.
• Meduri A, et al. Sci Rep. 2020;doi:10.1038/s41598-020-77194-9.
• Muyldermans A, et al. BMJ Open Ophthalmology. 2021;doi:10.136/bmjophth-2021-000733.
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• Sawant OB, et al. Ocul Surf. 2021;doi:10.1016/j.jtos.2020.11.002.
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• Wikramaratna PS, et al. Euro Surveill. 2020;doi:10.2807/1560-7917.ES.2020.25.50.2000568.
• Zeng W, et al. JAMA Ophthalmol. 2020;doi:10.1001/jamaophthalmol.2020.3906.

• For more information:
• Claudio Azzolini, MD, can be reached at Department of Medicine and Surgery, Università degli Studi dell’Insubria, Via Guicciardini 9, 21100, Varese, Italy; email: [email protected].
• Preeya K. Gupta, MD, can be reached at Duke University, Department of Ophthalmology, Box 3802, Durham, NC 27710; email: [email protected].
• Ashok Kumar, PhD, can be reached at Kresge Eye Institute, 4717 St. Antoine, K-416, Detroit, MI 48201; email: [email protected].
• Peter Raus, MD, PhD, can be reached at 130 Stationsstraat, 2440 Geel, Belgium; email: [email protected].

Click here to read the Point/Counter to this Cover Story.

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