Presence of SARS-CoV-2 on the conjunctival mucosa in patients hospitalized due to COVID-19: Pathophysiological considerations and therapeutic implications

Introduction: Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a worldwide pandemic, due to its great capacity to invade the human body. Previous studies have shown that the primary route of invasion of this virus is the human respiratory tract via the co-expression of ACE2 receptor and TMPRSS2, a serine protease on the cellular surface. Interestingly, this condition is present not only on the respiratory epithelium but on the conjunctival mucosa, as well. Thus, we hypothesized that SARS-CoV-2 is present on the conjunctival mucosa. Aim: To prove that SARS-CoV-2 can be detected in the conjunctiva. Methods: Previously naso-pharyngeal swab-sample based real-time polymerase chain reaction (PCR) positive COVID-19 infected patients were selected at the COVID Care Centers of Semmelweis University, Budapest, Hungary. The study was approved by the ethical committee of Semmelweis University. During their recovery, both nasopharyngeal and conjunctival swab-samples were taken and PCR method was used to detect the presence of SARS-CoV-2 RNA. Appropriate statistical analysis was performed. Results: The study population consisted of 97 patients, 49 females (50.5%) and 48 males (49.5%), with a mean age of 67.2 ± 11.9 years. During recovery, with nasopharyngeal swabs, the PCR test was positive in 55 cases (56.70%), whereas


INTRODUCTION The COVID-19 pandemic
Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the greatest worldwide pandemic since the Spanish flu in 1918-1919 [1]. It has greatly impacted the national and global healthcare systems and demanded over 4 million lives out of more than 190 million people infected [2]. The primary entrance location of SARS-CoV-2 infection is in the mucosal cells after being exposed to the virus directly or indirectly and facilitated by the binding of coronavirus spike protein subunit to the angiotensin-converting enzyme 2 (ACE2) receptor [3][4][5][6][7][8][9][10][11][12][13][14][15]. The cellular entry also requires the priming of the protein by a serine protease, TMPRSS2, or other proteases [11,15]. Thus, the co-expression of ACE2 and TMPRSS2 on the cellular surface are necessary for the cellular infection, a condition, which is present both on the epithelium of the respiratory tract and the conjunctival mucosa [11,15].
Pulmonary and extra-pulmonary manifestations COVID-19 is best known for its respiratory and systematic symptoms that manifest from asymptomatic infection to acute respiratory distress syndrome (ARDS) [7,15]. Classical symptoms are fever, cough, fatigue, weakness, loss of olfaction and taste [15,16]. In addition to the respiratory symptoms, this disease can affect other tissues and organs, such as hematologic-, cardiovascular-, gastrointestinal-, hepatobiliary-, endocrine-, dermatological-, neurological-and ophthalmological systems [15]. This latter one is not always appreciated and sometimes overlooked [17].

Previous ocular findings
As mentioned above, the conjunctival mucosa also can be exposed for SARS-CoV-2 infection, which than can elicit various ocular symptoms. The most common ocular manifestation of COVID-19 is conjunctivitis with epiphora, conjunctival congestion or chemosis, itching, foreign body sensation and/or watery discharge . Other pathologies and subjective symptoms as blurred vision, tarsal petechiae, hemorrhage or pseudomembrane, mucous filaments, anterior or posterior uveitis, retinal microangiopathy, microhemorrhages, cotton-wool dots and neuro-ophthalmologic manifestations such as diplopia, ophthalmoparesis and Miller-Fischer syndrome can also frequently occur . All of these underlines the importance of extending medical attention and care to the ocular system as well, during COVID-19 infection.

PATIENTS AND METHODS
The patients included in the present study provided an informed consent for the investigation, which was approved by the ethical committee of Semmelweis University. Patients had been previously admitted with SARS-CoV-2 positive nasopharyngeal swab to the COVID Care Centers of Semmelweis University, Budapest. During recovery, the conjunctival swab test was performed within 24 h after the control nasopharyngeal swab. There were 97 patients examined between January and April of 2021.
During recovery, both nasopharyngeal and conjunctival swabs were taken and analyzed with real-time polymerase chain reaction (PCR) in order to show the presence of SARS-CoV-2 RNA in the sample (in the Central Laboratory of Department of Laboratory Medicine, Semmelweis University). Conjunctival samples were taken from both eye (inferior conjunctival fornix) with synthetic fiber swabs and placed into the same test tube, meaning that infection on only one eye caused PCR positivity. Vital (body temperature, blood pressure, pulse, oxygen saturation, the amount of oxygen supplementation, respiratory rate) and laboratory parameters (CRP, PCT, IL-6, ferritin, blood count, renal and liver function, necro-enzymes and D-dimer) as well as symptoms (e.g., presence of fever or cough) were routinely recorded during the admission of the patient.
In some patients in the COVID-19 Centers, ophthalmological history was taken and basic examination was performed.

STATISTICS
Statistical analysis was performed using IBM ® SPSS ® Statistics for Windows, version 25.0 (IBM Corp., Armonk, N.Y., USA). All continuous variables were expressed as mean, median and interquartile ratio (IQR). Kolmogorov-Smirnov test was used to assess normality. Mann-Whitney test was used for comparison of continuous variables. Categorical variables were summarized as counts (percentages). Fisher's exact test was used for comparison of categorical variables. In all analysis, the level of significance was considered at P < 0.05.

RESULTS
The mean age of the patients was 67.22 ± 11.91 (n 5 97) years; 49 (50.52%) of them were females, 48 (49.48%) were males. The anamnestic, vital and laboratory parameters as well as symptoms of patients occurring are included in Table 1.
In several patients in the COVID-19 Centers, ophthalmologic history was taken and basic examination was performed, the findings are included in Table 2. Six (6.19%) patients presented ocular symptoms, such as foreign body sensation (1 patient), acute glaucoma (1 patient), strabismus (1 patient), exophthalmos (1 patient), ectropium (1 patient) and ocular discharge (2 patients). Two of these patients were positive for conjunctival SARS-CoV-2 PCR test, and both presented ocular discharge; that is, 25% of the patients with positive conjunctival swab had ocular symptoms. Every ocular finding that had not been presented in the anamnesis was considered a symptom. Fifteen patients (15.46%) had ophthalmologic anamnesis, such as glaucoma (1 patient   PCR positive conjunctival swab 12.50% had ocular symptoms and 50.00% of them had ophthalmologic anamnesis (Table 2). Patients were divided in two groups in two ways: 1) whether they had ocular symptoms revealed during the examination and 2) if their conjunctival swab tested positive for SARS-CoV-2 PCR test. We have found that the only statistically significant difference between the two groups of patients with and without ocular symptoms was the prevalence of ophthalmologic anamnesis, which was significantly higher in patients with ocular symptom(s) ( Table 1).

DISCUSSION
The salient finding of the present study is thatalthough only in few casesconjunctival tissue sample was positive for COVID-19 in the absence of nasopharyngeal positivity.
During the study of 97 hospitalized elderly patients (∼60 years old) with serious pulmonary and general COVID-19, symptoms were evaluated. Their majority required oxygen supplementation but none of them needed mechanical ventilation. In the present study 41 of all 97 patients required oxygen supplementation (42.3%).
At admission, the routine blood laboratory tests did not show significant difference between patients with or without ocular symptoms (Table 1). Also, there was no statistically significant difference between the vital and laboratory variables in patients with or without positive conjunctival SARS-CoV-2 PCR test result. Samples for PCR tests were taken from the conjunctival epithelium within 24 h after performing the control nasopharyngeal swab tests. Nasopharyngeal tissue sample showed positive SARS-CoV-2 PCR result in 55 cases (56.70%), whereas conjunctival swabs were positive in 8 cases (8.25%). The relatively low number of PCR positive nasopharyngeal swabs test is likely due to the fact that many patients were recovering from COVID-19 after 1-3 weeks of admission. Also, it is interesting to note that human conjunctival epithelium contains much less ACE2 receptors compared to the lower respiratory tract [11,15]. Perhaps because of this reason, conjunctival swab can be PCR negative in spite of being a potential entrance route for SARS-CoV-2 virus.
Although conjunctivitis can be a present in COVID-19, previous findings of Bertoli et al. indicated that the exact incidence of conjunctivitis in COVID-19 patients is unclear, ranging between 0.8% and 31.6% [7]. In 2020, Güemes-Villahoz et al. reported that about 12% of COVID-19 patients presented conjunctivitis symptoms, which were associated with the disease and usually occurred in the early phase of the disease [19]. Moreover, Wu et al. have shown that in 73.7% of patients hospitalized due to COVID-19 had positive RT-PCR test for COVID-19 from the nasopharyngeal swabs, and of these, 5.2% of them yielded positive tests for SARS-CoV-2 in their conjunctival as well as nasopharyngeal specimens. A total of 31.6% of their patients had ocular manifestations consistent with conjunctivitis, including conjunctival hyperemia, chemosis, epiphora, or increased secretions. Patients with ocular symptoms were more likely to have higher white blood cell and neutrophil counts and higher levels of procalcitonin, C-reactive protein, and lactate dehydrogenase than patients without ocular symptoms [3]. Feng et al. reported in 2021 that 9.5% of hospitalized COVID-19 patients exhibited ocular signs and symptoms, whereas Chen et al. found that 5.0% of their 535 studied patients presented with conjunctival congestion as the initial COVID-19 symptom [4]. In the present study 8.25% of the hospitalized patients were positive for conjunctival COVID-19 swab PCR test.

Physiology International 109 (2022) 4, 475-485
On the basis of previous experimental [29,30] and human [31] studies one can consider a secondary conjunctival infection due to spreading of infection along mucus membranes. Also, human studies have shown that ACE2 and other SARS-CoV-2 co-receptors are expressed in human ocular tissues [31] and that certain diseases can change protein expressions. This may be important, since our study demonstrated that ocular symptoms were present in patients whom had a history of ophthalmologic diseases, which can potentially change the expression of viral receptor proteins [31].
In hospital settings SARS-CoV-2 can be also transmitted by virus carrying stuff or patients derived respiratory droplets [32] reaching the conjunctiva of patients. In addition, SARS-CoV-2 RNA has been detected post mortem in conjunctival tissues and intravitreal biopsies [33]. Thus the original entry of virus cannot always be ascertained.
All in all, the previous and present findings suggest that the conjunctiva can be seriously affected by COVID-19, either directly by the virus or indirectly from systemic COVID diseases. Also, due to the special anatomical connection between the conjunctiva and nasal cavities, the ocular surface can be an entry route of nasopharyngeal COVID-19 infection, as the tear film is transported to the inferior nasal meatus by the nasolacrimal duct [8].

Limitation of the study
There are limitations of our pilot study. In the COVID Care Centers of Semmelweis University detailed ophthalmic assessment was not possible due to the personal protective equipment including compulsory face shield. Thus, we focused the investigation on laboratory parameters and the ocular anterior segment symptoms. In addition, we could investigate only limited number of cases, and no technical replicates were done. During the time of our investigation in early 2021, only limited amount of COVID vaccines was available in Hungary, mostly distributed among healthcare workers and elderly patients above the age of 80 years. Thus, the patients included in this study received none or only one dose of one of the COVID vaccines, mostly Pfizer, Moderna, AstraZeneca, Sinopharm or Sputnik, and because of that we did not record immunization history.

CONCLUSIONS
Although only in few cases, the data of the present study provides a proof of concept that SARS-CoV-2 can be present on the conjunctival mucosa even in nasopharyngeal negative patients, a finding, which can have clinical importance. Also, on the basis of this findings one can hypothesize thatin addition to the respiratory tractthe conjunctiva can be an entrance route for SARS-CoV-2 to the human body, which however needs to be substantiated by follow up studies. Nevertheless, in high risk conditions in addition to covering the mouth and nose with mask, the protection of the eyes is also strongly recommended.