The infection evidence of SARS-COV-2 in ocular …...2020/02/26 · The infection evidence of...

The infection evidence of SARS-COV-2 in ocular

surface： a single-center cross-sectional study

Xian Zhang1,*, Xuhui Chen1,*, Liwen Chen1, Chaohua Deng1, Xiaojing Zou2, Weiyong

Liu3, Huimin Yu1, Bo Chen1,#, Xufang Sun1,#

Affiliation:1Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, China2Department of Emergency, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, China3Department of Clinical Laboratory, Tongji Hospital, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, China.

*Co-authors:

Xian Zhang and Xuhui Chen contributed equally to the manuscript. Email:

[email protected], [email protected]

#Corresponding authors:

Bo Chen, E-Mail: [email protected]

Xufang Sun, E-Mail: [email protected]

Department of Ophthalmology, Tongji Hospital, Tongji medical collage, Huazhong

University of Science and technology, 1095 Jiefang Ave., Wuhan, China, 430030,

Funding Sources

This work was supported by grants from the National Natural Science Foundation of

P.R. China (Grant No. 8197033356).

All rights reserved. No reuse allowed without permission. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted February 26, 2020. ; https://doi.org/10.1101/2020.02.26.20027938doi: medRxiv preprint

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

https://doi.org/10.1101/2020.02.26.20027938

Purpose: The aim of this study was to identify whether SARS-COV-2 infected inocular surface.Methods: Cross-sectional study of patients presenting for who received a COVID-19

diagnosis, from December 30, 2019 to February 7, 2020, at Tongji hospital, Tongji

medical college, Huazhong University of Science and Technology. Demographics,

temperature was recorded, blood routine test (Rt), chest Computed Tomography (CT)

were took intermittently, and SARS-COV-2 real-time

reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay were arranged for

the nasopharyngeal and conjunctival swab samples.

Results: A total of 102 patients (48 Male [50%] and 54 Female [50%]) with clinical

symptoms, Rt, and chest Computed Tomography (CT) abnormalities were identified

with a clinical diagnosis of COVID-19. Patients had a mean [SD] gestational age of

57.63 [14.90] years. Of a total of 102 patients identified, 72 patients (36 men [50%]

and 36 women [50%]; mean [SD] age, 58.68 [14.81] years) confirmed by laboratory

diagnosis with SARS-COV-2 RT-PCR assay. Only two patients (2.78%) with

conjunctivitis was identified from 72 patients with a laboratory confirmed COVID-19.

However, SARS-COV-2 RNA fragments was found in ocular discharges by

SARS-COV-2 RT-PCR only in one patient with conjunctivitis.

Conclusions: Although we suspect the incidence of SARS-COV-2 infection through

the ocular surface is extremely low, the nosocomial infection of SARS-CoV-2 through

the eyes after occupational exposure is a potential route. The inefficient diagnostic

method and the sampling time lag may contribute to the lower positive rate of

conjunctival swab samples of SARS-COV-2. Therefore, to lower the SARS-COV-2

nosocomial infection, the protective goggles should be wore in all the health care

workers.



https://doi.org/10.1101/2020.02.26.20027938

Introduction

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2)

associated with severe human infected disease (COVID-19) outbroke starting from

Wuhan city, in China.1 Most recently, Wang D et al reported 138 hospitalized

SARS-COV-2 pneumonia cases with a hospital-associated transmission rate of 41%,

among whom 70% were medical staffs.2 To date, nearly 80, 000 people have been

infected by COVID-19, including over 3000 medical staffs. Therefore, in order to

curb the spread of SARS-COV-2, and reduce the nosocomial infection, the exact

routes of transmission need to be further studied and confirmed.

Although primarily affecting the respiratory tract, it also involves extra-pulmonary

sites, including the digestive tract and other organs.3 SARS-CoV-2 infected patients

develop respiratory illness, with the first symptoms of fever, cough and fatigue that

quickly progress to pneumonia. COVID-19 were also contributed to extra-pulmonary

manifestations in a number of patients at the onset of the illness, such as headache,

diarrhoea, nausea and vomiting,4 or even presented with asymptomatic infection.8

Despite the intensive work that has been focused on an understanding of the

transmission of SARS-CoV-2, the spread mode of the SARS-CoV-2 is unclear.

To our knowledge, limited investigations have been conducted to date into the clinical

features of SARS-COV-2 in ocular surface. A few of COVID-19 patients with

conjunctivitis, or even as the first symptom, make the early diagnosis and effective

protection a great difficulty. A recent research has demonstrated that SARS-COV-2

can be detected in the conjunctival sac of patients with COVID-19 patients or

suspected.5 However, none of these patients had ocular symptoms. We therefore

conducted a study to describe the clinical spectrum of ocular symptoms and

laboratory test in conjunctival swab samples, we found a rare case of nosocomial

SARS-COV-2 infection with conjunctivitis in a nurse, which suggests that ocular

transmission may be a potential route of nosocomial transmission of the

SARS-COV-2.



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Methods

A Cross-sectional study was conducted of all patients who received a COVID-19

diagnosis between December 30 30, 2020, and February 7, 2020, at Tongji hospital,

Tongji medical college, Huazhong University of Science and Technology. The aim of

this study was to identify whether ocular surface is an infected target of SARS-CoV-2.

This study was approved by the ethics committee of Tongji hospital and was

performed in accordance with the tenets of the Declaration of Helsinki.6

Clinical specimens for 2019-nCoV diagnostic testing were obtained in accordance

with CDC guidelines. To diagnosis the COVID-19, blood routine test (Rt), chest

Computed Tomography (CT) and SARS-COV-2 real-time

reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay were arranged.

Serum was collected in a serum separator tube and then centrifuged in accordance

with CDC guidelines. Laboratory-confirmed cases were identified with the criteria of

at least one positive result from a respiratory specimen using RT-PCR assays. All

patients invited to participate in the study provided consent for the nasopharyngeal

and conjunctival swab samples with synthetic fiber swabs.

Then, these swab samples were detected via real-time RT-PCR assays as previously

described.7 Briefly, RNA extraction from nasopharyngeal swabs and conjunctival

swabs was performed RT-PCR on a Real Time PCR System. Thermal cycling was

initiated with an initial incubation at 50°C for 15 min, 95°C for 3 min. Each PCR

cycle consisted of heating at 95°C for 15 s for melting and at 60°C for 30 s for

annealing and extension. The primers and probes for SARS-CoV-2 detection were

selected according to the National Pathogen Resource Center (National Institute for

Viral Disease Control and Prevention, China CDC), and the sequences were as

follows7:

forward primer 5′-ACTTCTTTTTCTTGCTTTCGTGGT-3′;

reverse primer 5′-GCAGCAGTACGCACACAATC-3′;

and the probe 5′CY5-CTAGTTACACTAGCCATCCTTACTGC-3′BHQ1.

The statistical analysis was performed using SPSS software version 22.0 (SPSS Inc,



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Chicago, IL). The descriptive data were presented as percentage, mean, and standard

deviation. All the symptoms and examination results are present in the Powerpiont

2016.

Results

A total of 102 patients (48 Male [50%] and 54 Female [50%]) with clinical symptoms,

Rt, and CT abnormalities were identified with diagnosis of COVID-19. Patients had a

mean [SD] gestational age of 57.63 [14.90] years. Of a total of 102 patients identified,

72 patients (36 men [50%] and 36 women [50%]; mean [SD] age, 58.68 [14.81] years)

confirmed by laboratory diagnosis. The time for conjunctival sampling were had a

mean [SD] day of 18.15 [7.57] days. Demographic data including patients’ age,

gender and the time for conjunctival sampling were shown in Fig. 1.

Only two patients (2.78%) with conjunctivitis were identified from 72 patients with a

laboratory confirmed COVID-19. However, SARS-CoV-2 was found in ocular

discharges by RT-PCR only in one patient, the symptoms and laboratory test were

shown in Fig. 2. Briefly, a 29-year-old nurse working in the Emergency Department

at Tongji hospital, Wuhan City, China was referred to the Department of

Ophthalmology at Tongji Hospital on February 1st, 2020 due to excessive tearing and

redness in both eyes (Fig. 3A). No other systemic symptoms except for a moderate

fever of 38.2℃ was reported on January 31st, 2020. The ocular examination revealed

conjunctival congestion and watery discharges in both eyes with normal best

corrected visual acuity, normal corneal epithelium, quiescent anterior chamber and no

tenderness or enlargement of the preauricular lymph node. We therefore excluded the

possibility of conventional conjunctivitis, such as bacterial conjunctivitis,

hemorrhagic conjunctivitis, allergic conjunctivitis, according to her clinical symptom

and sign. She clarified medical N95 respirators continuously wore during operation,

while occasionally worked with a dislocated eye mask touching her eyelids.

Considering the occupational exposure by SARS-COV-2, SARS-COV-2 related assay

were arranged. Surprising, the chest CT showed multiple peripheral ground-glass

opacities in both lungs (Fig. 3D-F). The conjunctival and oropharyngeal swabs tested



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for SARS-COV-2 were both positive. The blood Rt showed a normal total white blood

cells with elevated monocyte counts. On the basis of her epidemiologic characteristics,

clinical manifestations, chest images, and laboratory findings, this patient was

diagnosed with SARS-COV-2 infected acute viral conjunctivitis and pneumonia.

Before admission, the patient reported persistent a 4-day history of conjunctivitis and

a 3-day history of fever. Ganciclovir eye drops was used to control her conjunctivitis,

the patient’s conjunctivitis vital signs remained stable during her home quarantine,

apart from the development of intermittent fevers, accompanied by periods of cough

from day 4 to 9 post of illness (Fig. 2). Although, SARS-COV-2 RT-PCR assay for the

nasopharyngeal and conjunctival swab were negative 5 days after the conjunctivitis

taken a turn for the better, a fever reappear, and the Chest Radiographs showed that

her pneumonia aggravated at day 10 post illness. Then, she was arranged to the

hospital treated according to the guidance of CDC, and then discharged 11 days post

hospitalization, accompanied with a slight cough.

Fig.1 Demographic data: Scatter plot of the patients' distribution characteristicsaccording to the age, gender and sampling date from the date of onset. Thesampling date of conjunctival swab varied from the 6th day to the 46th day, withan average of 18.15 days.(red spots: females; blue spots: males)



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Fig. 3 Clinical photographs of the positive patient regarding conjunctivitis andchest CT: (A) binocular conjunctival congestion which is a classic sign for viralconjunctivitis; (B,C) conjunctivitis completely subsided and did not recur 4days after onset; (D-F) dynamic changes of peripheral ground-glass opacities inboth lungs (yellow arrows) which illuminate the pneumonia caused bySARS-COV-2 infection.The pulmonary lesions were aggravated on the 10thday, and the lesions were basically absorbed on the 18th day.

Fig.2 Maximum Body Temperatures, Symptoms, and Laboratory Tests ofPositive Patient according to Day of Illness and Day of Hospitalization,January 31 to February 20, 2020. The early symtems were fever andconjunctivitis which last for 4 days. Both oropharyngeal and conjunctivalswabs PCR were positive on the 3rd day followed by 3 negative tests. (oPCR:oropharyngeal swabs PCR; cPCR: conjunctival swabs PCR; Lym: Peripheral BloodLymphocyte Count; EMD: Emergency Department; Oph: Ophthalmology Department.)



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DiscussionLike other highly contagious respiratory viruses, respiratory droplets are considered

as the main route of SARS-COV-2 transmission.8 However, other possible

transmission ways should be taken into account owing to the rapid transmission. A

recent bioinformatics analysis study analyzed 4 datasets with single-cell

transcriptomes of lung, oesophagus, gastric, ileum and colon, and SARS-CoV-2 was

found in stool samples of patients with abdominal symptoms.9 Moreover, Lofy KH et

al10 detected the SARS-COV2 in a patient's stool, which warns us of the risk of fecal

oral transmission. Similarly, SARS-CoV-2 was also directly detected in the

oesophageal erosion and bleeding site in a case with severe peptic ulcer symptom

reported by Zhong et al.11 Therefore, the real spread route is still a mystery, other

transmitted ways should be further studied and confirmed.

In the current study, we found that two patients (2.78%) with conjunctivitis was

identified from 72 patients with a laboratory confirmed COVID-19. However,

SARS-CoV-2 was found in ocular discharges by RT-PCR only in one COVID-19

patient. Although the incidence of conjunctivitis is extremely low, these results

demonstrated that SARS-CoV-2 have shown a capacity to use the eye as a portal of

entry and cause ocular disease. To our knowledge, several anatomical and mucosal

immune properties permitted the eye as both a potential site of virus infected site as

well as a gateway for respiratory infection.12-13 In coincidence with our result, SARS-

CoV was detected by RT-PCR in tear samples from three probable cases.14 Resemble

as SARS-CoV, entry of SARS-CoV-2 via the host functional receptor is mediated by

ACE2.15 Moreover, Sun and colleagues16 found that ACE2 expressed in human cornea

and conjunctival tissues, which providing strong evidence for our diagnosis.

Furthermore, Dr. Guangfa Wang, a member of the national expert panel on pneumonia,

reported that he was infected by SARS-COV-2 during the inspection in Wuhan,

through unprotected eye exposure.17 Similarly, an anesthesiologist with insufficient

eye protection confirmed with COVID-19 and also presented conjunctivitis as the

initial symptom. The laboratory test revealed that the nasopharyngeal swab was

positive while the conjunctival swab was negative.5 On the contrary, our case



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initially presented similar conjunctivitis with a positive conjunctival PCR result.

Considering the same occupational exposure of Dr. Wang, the anesthesiologist and the

nurse with positive conjunctival PCR result in our case, and the SARS-COV-2

infected mechanism, we believe that ocular transmission of SARS-CoV-2 must be

seriously considered, especially in health care workers.

The negative results of conjunctival sac in other 71 patients, may be owing to the

lower viral concentration, the sampling time lag, and the lower positive rate of the

inefficient diagnostic method. Ziad and colleague18 found that tracheal aspirates

yielded significantly higher SARS-CoV loads, compared with the nasopharyngeal

swab and sputum specimens. This suggests that the viral concentration and genome

fraction is diverse in different sites. In consideration of the ocular surface is an open

microenvironment, and the viral may transport to the inferior meatus of the nose

rapidly,19 the SARS-CoV-2 concentration in ocular surface is likely to be very low.

We also found that suspected patients often had 2~3 repeated tests of nasopharyngeal

swabs before the positive result confirmed SARS-CoV-2 infection. The sampling time

lag and the inefficient diagnostic method may contribute to this lower positive rate of

SARS-CoV-2. de Wit and colleagues20 demonstrated that, in the rhesus macaque

model, MERS-CoV RNA could detect in the conjunctiva, and the viral loads could no

longer be detected in the conjunctiva 6 days post infection. However, the mean time

for conjunctival sampling are 18.15 days in our present study. Another thing needs to

be considered is that the lower positive rate of RT-PCR makes early diagnosis of

SARS-CoV-2 a challenge. Therefore, improvements in the sensitivity of molecular

diagnostic methods need to be taking in the future.

There are several limitations need to be considered. First, the conjunctival scraping

test should be done as early as possible if we find ocular symptoms in a suspected one.

The mean time for conjunctival sampling in our study are 18.15 days, in which

exceeded the optimal detection time. Second, how to increase the positive rate. The

most commonly used chest CT examination has certain limitations for the special

suspected groups, such as pregnant women. In addition, we found that 2~3 repeated

tests did in suspected patients of nasopharyngeal swabs before the SARS-CoV-2 was



https://doi.org/10.1101/2020.02.26.20027938

obtained, which may be related to the false-negative result of RT-PCR. Therefore, the

low viral load in the ocular surface and the lower positive rate of RT-PCR makes early

diagnosis of SARS-CoV-2 a challenge. Recently, Doan21 reported that the influenza

virus and rubella virus found in patients’ conjunctival sac or tears by the next

generation sequencing rapidly, which provides us with a feasible direction for future

study.

Conclusions

In conclusion, we suspect the incidence of SARS-COV-2 infection through the ocular

surface is extremely low in the general population, however, considering the common

feature of the positive cases which are occupational exposure accompanied with

conjunctivitis in the early stage, the higher viral aerosol load in the hospital and more

opportunities to contact with COVID-19 patients, we highlight that ocular

transmission is a potential important way of occupational exposure for medical staff.

Therefore, To lower the risk of SARS-COV-2 nosocomial infection, the protective

goggles should be wore in all the health care workers, especially who work in the

Fever Outpatient and Infection Wards. Patients with conjunctivitis in the epidemic

area should also be treated seriously to rule out the COVID-19. Moreover, to date, the

lower positive rate of conjunctival sac may be ascribed to the lower viral

concentration, the sampling time lag, and the inefficient detection methods. Our study

highlights the need for further research into the efficient detection methods of

SARS-COV-2.

Declaration of competing interest

All authors report no relevant conflicts of interest, financial or otherwise.

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