Tear Film SARS-nCoV-2 Detection in Symptomatic and Pauci-symptomatic
Conditions: Covid19, Eye Diseases
Sponsor: Universitaire Ziekenhuizen KU Leuven

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 1 van 23 
 
 
 
[L1] 
 
 
Protocol Full Title:  
Tear film SARS-nCoV-2 detection in symptomatic and  pauci-symptomatic patients. 
 
Protocol Acronym/short title: 
Eye-COVID 
 
Version and date of protocol: 
V8.0 dd 16-09-2020 
 
Principal Investigator: 
Name: dr. Heleen Delbeke 
Address: UZ Leuven campus Gasthuisberg, Ophthalmology Department, Herestraat 49, 3000 
Leuven 
Telephone: +32 16/ 33 29 20 
Email: heleen.delbeke@uzleuven.be 
 
Contact: 
Name: dr. Laura Leysen 
Address: UZ Leuven campus Gasthuisberg, Ophthalmology Department, Herestraat 49, 3000 
Leuven 
Telephone: +32 16/ 34 61 09 
Email: laura.leysen@uzleuven.be 
 
Signature Principal Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Heleen Delbeke MD 
 
 
 
 
 
Date: 16-09-2020 
Principal Investigator 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 2 van 23 
 
 
 
Protocol approved by 
 
 
Prof. dr. Ingele Casteels MD PhD 
Head of the Department of Ophthalmology 
(Sub Investigator) 
 
Dr. Pieter-Paul Schauwvlieghe, MD  
(Sub Investigator) 
 
 
Dr. Isabelle Saelens MD PhD 
(Sub Investigator) 
 
Dr. Karel Van Keer MD PhD 
(Sub Investigator) 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 3 van 23 
 
 
 
Apotheker klinisch bioloog Stefanie Desmet 
(Sub Investigator) 
 
 
 
 
Prof. Marie Joossens PhD 
(Counceling Sub Investigator) 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 4 van 23 
 
 
 
 
Title of clinical trial 
 
 
Tear film SARS-nCoV-2 detection in symptomatic 
and pauci-symptomatic patients. 
 
Protocol Short Title/Acronym 
 
 
Eye-COVID 
 
Funding 
 
 
 
R95328 
 
Principal Investigator 
 
 
Dr. H. Delbeke 
Sponsor 
 
University Hospitals Leuven 
Herestraat 49 
3000 Leuven 
Sponsor Reference Number  
 
S 64002 
 
Purpose of clinical trial 
 
 
 
To investigate the presence of SARS-nCoV-2 in 
the tear film of symptomatic and pauci-
symptomatic SARS-nCoV-2 positive patients. 
 
Trial Design 
 
 
Exploratory study using biological samples    
Summary of eligibility criteria 
 
 
• 
≥ 18 years old. 
• 
Willing to undergo sampling of the 
conjunctiva.  
• 
Willing to fill in a questionnaire.  
• 
Fluent in written and verbal Dutch.  
• 
Capable of giving informed consent.  
• 
Part 1 only: positive test for SARS-nCoV-2 
on a nasopharyngeal swab.  

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 5 van 23 
 
 
 
• 
Part 2 only: mild symptoms of COVID-19 
including symptoms of upper or lower 
respiratory tract infection and any body 
temperature elevation (criteria 
Sciensano for testing patients and 
healthcare workers (April 12 2020)) 
without the need for hospitalization.  
 
 
Version and date of final protocol 
 
 
 
V8.0 dd 16-09-2020 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 6 van 23 
 
 
 
Table of contents  
 
1. 
Background, rationale and novelty .................................................................................................................. 7 
2. 
Trial objectives and design .............................................................................................................................. 9 
2.1 Preparation, organization and management of this exploratory study ...................................................... 10 
3. 
Selection and withdrawal of subjects ............................................................................................................ 12 
3.1 Inclusion criteria .......................................................................................................................................... 12 
3.2 Exclusion criteria ......................................................................................................................................... 12 
3.3 Expected duration of trial ........................................................................................................................... 12 
4. 
Trial procedures ............................................................................................................................................ 13 
4.1 Summary of visits ........................................................................................................................................ 13 
4.2 Supervision and responsibilities .................................................................................................................. 13 
Insurance ....................................................................................................................................................... 13 
Informed consent .......................................................................................................................................... 14 
5. 
Assessment of efficacy .................................................................................................................................. 14 
6. 
Assessment of safety ..................................................................................................................................... 14 
6.1 Recording of adverse events ....................................................................................................................... 14 
6.2 Reporting to the Ethics Committee ............................................................................................................. 15 
7. 
Statistics ........................................................................................................................................................ 15 
7.1 Sample size .................................................................................................................................................. 15 
7.2 Analysis ........................................................................................................................................................ 15 
8. 
Quality assurance .......................................................................................................................................... 16 
9. 
Direct access to source data and documents.................................................................................................. 16 
10. Ethics and regulatory approvals .................................................................................................................... 16 
11. Data Handling ............................................................................................................................................... 16 
12. Data Management ......................................................................................................................................... 17 
13. Publication Policy ......................................................................................................................................... 17 
14. Financial Aspects .......................................................................................................................................... 17 
15. References ..................................................................................................................................................... 18 
16. Addenda ........................................................................................................................................................ 19 
Addendum A: Questionnaire to be filled in by all subjects ............................................................................... 19 
Addendum B: Explanation of the sampling ....................................................................................................... 21 
Addendum C: Poster/flyer for the recruitment ................................................................................................ 23 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 7 van 23 
 
 
 
1. Background, rationale and novelty 
 
A novel coronavirus, severe acute respiratory syndrome new coronavirus 2 (SARS-nCoV-
2) was first described early December 2019 (Wuhan, China) (1).  Since these first reports, 
the virus has been spreading and causing disease around the globe. Since the 11th of 
March 2020, the World Health Organization (WHO) declared the disease as a pandemic 
(2).  
This corona virus disease (COVID-19) causes flu-like symptoms (fever, myalgia, fatigue 
and cough), which can evolve to a severe bilateral pneumonia with significant dyspnea 
and chest pains. A subset of patients further deteriorates to an acute respiratory distress 
syndrome with the need for intensive care unit (ICU) administration and oxygen therapy. 
COVID-19 is a contagious infection spread by droplets and aerosols (3). Besides the flu-
like symptoms, certain publications report the presence of anosmia as well as ageusia as 
one of the first presenting symptoms of COVID-19 (4).  
This is consistent with the Google Trends analysis. The most common coronavirus related 
search queries are loss of smell, fever, and chills. While nasal congestion and diarrhea 
were fifth and sixth on the search terms. It is the fourth most searched term - eye pain - 
that has now caught the eye (quite literally) of researchers as it could be a possible 
symptom of the COVID 19 (5). 
Independent of the severity of respiratory symptoms, the presence of the virus in tears 
(positive for SARS-nCoV-2 via qPCR assessment) has been described in patients with 
conjunctivitis (6,7). This in line with earlier results during the SARS epidemic of 2003 (8). 
However, the presence of SARS-nCoV-2 in tears, implies that the disease might possibly 
be transmitted via this body fluid, especially during certain specific procedures such as 
pneumotonometry and excimer refractive laser surgery. Specific to these procedures is 
the use of a jet of air and a laser beam, transforming tears to small droplets (9–11).  
To further investigate the potential spread of the virus through tears, we aim to set up a 
two-part study. The first part of our study is to confirm the detectability of the SARS-
nCoV-2 in the tear film of symptomatic patients. Patients admitted at our University 
Hospital Leuven, Belgium with a positive nasopharyngeal swab for SARS-nCoV-2 will be 
informed about our project. They will be asked to sign an informed consent for serial 
swabbing of the conjunctiva every 3 days until the end of their hospitalization. 
A conjunctival swab is chosen above other methods such as Schirmer’s test since a swab 
is besides faster also easier to teach and requires less contact with the volunteer. The 
serial swabbing will not only be informative on the duration of viral shedding in the tears, 
but also on the timing that SARS-nCoV-2 becomes detectable with qPCR in the tear film. 
Furthermore, a questionnaire (cf. Addendum A) will be used to search for correlations 
between SARS-nCoV-2 positive conjunctival swabs and the patient’s disease stage 
(including results of routine blood samples), presence of ocular symptoms and/or 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 8 van 23 
 
 
 
perception of anosmia and/or ageusia. Especially this anosmia part might be interesting. 
A correlation between anosmia and a positive PCR in the tear film is suspected based on 
the connection between the nose and eyes via the nasolacrimal duct and the proximity 
of the olfactory nerve to the orbital cavity and eye.  
The other  parts of our study aims to confirm the detectability of the SARS-nCoV-2 virus 
in the tear film of pauci-symptomatic volunteers, more specifically patients presenting at 
the emergency room of the University Hospitals Leuven, health care workers with mild 
symptoms and patients admitted for other reasons though SARS-nCoV-2 positive. 
Presumably, the rate of pauci-symptomatic carriers is higher within health care workers 
compared to the general population. Emergency patients and health care workers are 
preferred over pauci-symptomatic pre-operative patients, since prevalence of SARS-
nCoV-2 is low in these pre-operative patients (data UZ Leuven Laboratory Medicine (1 
June 2020): prevalence of around 0.2%). Similar to the symptomatic patients, all 
volunteers will be informed about our project and asked to sign an informed consent for 
simultaneous swabbing of the conjunctiva and the nasopharynx. The pauci-symptomatic 
group will include only volunteers already listed for nasopharyngeal swabbing based on 
their symptoms (to avoid unnecessary use of nasopharyngeal swabs). By testing pauci-
symptomatic volunteers, we intend to better substantiate current guidelines in 
ophthalmology practice. Mostly “deemed-healthy” patients visit outpatient clinics, but 
when carrying the virus, they are the highest risk of contaminating health care workers. 
Proving presence of (viable) SARS-nCoV-2 virus in the tear film of pauci-symptomatic 
volunteers might change our ophthalmology practice in the long run. Furthermore, we 
want to compare results of both the ocular and nasopharyngeal swab (both positive for 
SARS-nCoV-2; both negative for SARS-nCoV-2; discordant results, with either eye or 
nasopharynx positive and the corresponding sample negative for SARS-nCoV-2). For 
patients with discordant results, we purpose to identify potential correlations between 
disease-severity and detection of SARS-nCoV-2 in different body fluids by using the same 
questionnaire mentioned above (cf. Addendum A). The difference in RNA viral load (by 
means of quantitative result (Cycle threshold (Ct) of the SARS-CoV-2 PCR) in the tears of 
different patients at different moments in time (i.e. the assessment of the evolution of 
quantitative result) will be indicative for the transmissibility of the virus. However, to 
evaluate genuine transmissibility, swabs positive for SARS-CoV-2 via qPCR will be sent to 
the REGA institute (i.e. the Laboratory of Piet Maes) for viral culture and detection of 
subgenomic (sg) RNA. This is in contrast to most previously published studies focusing 
only on the detection of SARS-CoV-2 RNA by PCR. SARS-CoV-2 PCR is not able to 
differentiate between live or dead virus (6–8). It is important to make that difference in 
order to draw conclusions on potential transmission of the virus by tears. Detection by 
culture indicates if viable virus is present in the sample. Detection of sgRNA is an 
indicator for replication of the virus. The viability of the virus collected from tear samples 
will determine the risk of spreading the virus via tears. 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 9 van 23 
 
 
 
This research project intends to provide a more thorough understanding of the presence 
of this new virus in the tear film of SARS-nCoV-2 positive patients. Furthermore, it could 
provide insight in the timing of the contagiousness of tears and highlights correlations 
with other signs and symptoms.  
 
 
Figure 1: study design.  
2. Trial objectives and design 
 
Primary outcome measures 
1) The presence of SARS-nCoV-2 in the tear film of symptomatic and pauci-symptomatic 
SARS-nCoV-2 positive patients.  
2) Correlations between SARS-nCoV-2 in the tear film and mild (like anosmia, ageusia, 
upper and lower respiratory tract infection) or severe symptoms (as pneumonia, need 
for hospitalization) of COVID-19.  
Secondary outcome measures 
1) The stage of disease when SARS-nCoV-2 becomes detectable in the tears. 
2) Duration of shedding of SARS-nCoV-2 RNA in the tears. 
3) Difference in RNA viral load (by means of quantitative result (Cycle threshold (Ct) of 
the SARS-nCoV-2 PCR) in tears of different patients at different moments in time in order 
to estimate potential difference in transmissibility of the virus by tears. 
4) Assessment of evolution of quantitative result (Cycle threshold (Ct)) of the SARS-nCoV-
2 PCR over time within conjunctival SARS-nCoV-2 PCR positive patients 
5) Searching for a correlation between both quantitative and qualitative results of SARS-
nCoV-2 PCR on the nasopharyngeal and conjunctival swab (both positive for SARS-nCoV-
2; both negative for SARS-nCoV-2; discordant results, with either eye or nasopharynx 
positive and the corresponding sample negative for SARS-nCoV-2).  
EYE-COVID
STUDY 
Group 1: 
Symptomatic 
Serial sampling: 190 
patients 
Group 2: Pauci-
symptomatic 
Single sampling: 190 
patients

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 10 van 23 
 
 
 
6) Exploring the possibility of testing the tear film on SARS-nCoV-2 as a screening tool for 
early disease. 
7) Exploring the replication of the SARS-nCoV-2 virus, by differentiating between death 
and alive SARS-nCoV-2 virus through sg RNA-testing and tissue culture.  
 
2.1 Preparation, organization and management of this exploratory study 
 
Part 1: Serial sampling of symptomatic and confirmed positive patients 
Patients will be informed about the study by a health care worker at a non-ICU unit 
(“rampen unit”) of the University Hospitals Leuven, Belgium.  
In case a candidate is interested in participating and tested positive for SARS-nCoV-2 on 
his/her nasopharyngeal swab, he/she will be informed about the study goals by the same 
health care worker. The volunteer will receive an information letter and a copy of the 
informed consent to take home.  
Study procedure 
After signing the informed consent, each patient will be asked to fill in a questionnaire 
(cfr. Addendum A). 
The simplicity of performing a conjunctival swab makes it possible to implement in the 
daily routine of health care workers, avoiding additional waste of personal protective 
equipment.  
Conjunctival sampling will be performed on both eyes after the application of a drop of 
topical anesthesia (Minims® Oxybuprocaine Hydrochloride 0.4%, Bausch and Lomb, 
Aubenas, Frankrijk). A timed 5 minutes will be in between application of the drop and 
the sampling. The sampling will be performed by a trained nurse and/or medical student 
and/or resident after proper training of correct sampling (cfr Addendum B). 
Each eye will be sampled with a single, sterile, nylon, flocked swab (FLOQSwabs®; Copan, 
Brescia, Italy). This FLOQSwabs® is a different swab than the one used for routine 
nasopharynx SARS-nCoV-2 testing. 
The sampling will be done from the nasal to temporal inferior conjunctival sac, the swab 
will be swirled in the opposite direction of the sampling itself.  
The swabs will be placed in a tube with universal transport medium (UTM) and 
transferred to a −80°C freezer until further processing. 
The conjunctival swab will be tested for SARS-nCoV-2 presence by making use of in-
house reverse transcription real-time polymerase chain reaction (RT-PCR) designed for 
routine diagnostics targeting specific SARS-nCoV-2 genes in the clinical laboratory of the 
University Hospitals Leuven or at the REGA institute (laboratory of Dirk Daelemans). 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 11 van 23 
 
 
 
Serial sampling 
Sampling will be repeated every 3 days to evaluate if PCR becomes positive during the 
course of disease and to monitor the duration of detectability of the virus in tears. 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 12 van 23 
 
 
 
Part 2: Single sampling of pauci – symptomatic patients 
Emergency patients, health care workers and patients hospitalized for a different 
condition/reason with a limited amount of symptoms (cfr. Inclusion criteria) that apply 
for a nasopharyngeal swab to confirm COVID-19 will be informed about the study by a 
pre-indicated health care worker.  
When the patient is interested in participating he/she will be informed about the study 
goals by the same health care worker. The volunteer will receive an information letter 
and a copy of the informed consent to take home. 
After signing the informed consent, each patient will be asked to fill in a questionnaire 
(cfr. Addendum A). 
Study procedure 
The conjunctival swabbing has been explained in detail in part 1.  
3. Selection and withdrawal of subjects 
 
3.1 Inclusion criteria 
 
• Subject needs to be above 18 years old. 
• The subject is willing to undergo sampling of the conjunctiva.  
• The subject is willing to fill in a questionnaire.  
• The subject is fluent in written and verbal Dutch.  
• The subject is capable of giving informed consent.  
• Applicable for part 1 only: the subject test positive for SARS-nCoV-2 on a 
nasopharyngeal swab. The time window between a positive nasopharyngeal swab 
and the first conjunctival swab may be no more than 3 days. 
• Applicable for part 2 only: mild symptoms of COVID-19 including symptoms of upper 
or lower respiratory tract infection and/or any body temperature elevation (criteria 
Sciensano for testing healthcare workers (April 12 2020)) without the need for 
hospitalization.  
 
3.2 Exclusion criteria 
 
• Allergy to Oxybuprocainehydrochloride 
• Pregnancy or lactation 
 
3.3 Expected duration of trial 
 
Inclusion is planned between the end of April till the end of April 2021.  

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 13 van 23 
 
 
 
4. Trial procedures 
 
4.1 Summary of visits 
 
Part 1: Serial sampling of symptomatic patients 
Sampling will take place at the “rampen unit” of the University Hospitals Leuven, 
Belgium. Once the patient is admitted and tested positive for SARS-nCoV-2 on 
nasopharyngeal swab, conjunctival sampling will take place every three days until the 
day of discharge. The time window between a positive nasopharyngeal swab and the 
first conjunctival swab may be no more than 3 days.  
Part 2: Single sampling of pauci-symptomatic patients 
Sampling will take place at the emergency department of the University Hospitals 
Leuven, Belgium by the swab team. The sampling will not be repeated.  
 
4.2 Supervision and responsibilities 
 
The study will be performed under the supervision of dr. H. Delbeke. The subjects will be 
recruited at the equivalent of the former “rampen unit”, the emergency department and 
via a flyer (cfr. Addendum C) at several outpatient clinics of the University Hospitals 
Leuven, Belgium and by making an announcement on the intranet webpage of the 
University Hospitals Leuven, Belgium. The sampling of the conjunctiva will be performed 
by a trained nurse and/ or medical student and/or resident (cfr. Addendum B). 
Volunteers will receive information about the study. Sampling will only occur upon 
written informed consent of the participants. The participating investigators will perform 
their part of the study fully in accordance with the terms of the Protocol, the applicable 
national and international laws (amongst others: the European General Data Protection 
Regulation of May 25, 2018) and apply and adhere to regulations and rules as, amongst 
others, the Declaration of Helsinki (2008) and ICH GCP Guidelines. Prof. M. Joossens is 
assigned as a free of charge consulting sub-investigator without the power to recruit 
patients.  
 
Insurance  
In accordance with the Belgian Law relating to experiments on human persons dated 
May 7, 2004, UZ/KU Leuven shall assume, even without fault, the responsibility of any 
damages incurred by a Study Patient from the UZ/KU Leuven site and linked directly or 
indirectly to the participation to the Study, and shall provide compensation therefore 
through their insurance (Amlin Corporate Insurance, polisnr. 299.053.700, Vanbreda Risk 
& Benefits, Plantin en Moretuslei 297, 2140 Antwerp).  
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 14 van 23 
 
 
 
Informed consent  
The Participating Site acknowledges that the Study can and will be conducted only on the 
basis of prior informed consent by the Subjects to participate in the Study. The 
Participating Site shall obtain a signed informed consent form (ICF) for all patients prior 
to their enrollment and participation in the Study in compliance with all applicable laws, 
regulations and the approval of the local Ethics Committee, if required. The Participating 
Site shall retain such ICFs in accordance with the requirements of all applicable 
regulatory agencies and laws. 
5. Assessment of efficacy 
 
Not applicable.  
6. Assessment of safety 
 
Sampling with topical anesthesia can cause a transient stinging and blurring of the vision 
after instillation. When used in larger amounts, epitheliopathy of the cornea can be 
noticed. This is rare in the case of only one drop. Systemic absorption will be reduced by 
asking the patient to gently close the lids and to press on the medial canthus for a 
minute following the instillation of the drops. There are no known interactions with 
other medical products. It will not be used during pregnancy or lactation, nor will it be 
used in patients with a known hypersensitivity to the product.  
After sampling the conjunctiva, there is a small possibility of a minor bleeding in the 
conjunctival sac which can induce a feeling of bruising of the tissue. Scarring due to the 
intervention is extremely rare (<0.01%). 
 
6.1 Recording of adverse events 
 
The risks associated with the study-specific intervention, i.c. the conjunctival sampling 
and the use of topical anesthesia are listed above (6. Assessment of safety). 
The participant will be asked to report any adverse event related to the study-specific 
intervention to the study team. These reported events will be documented by the 
Investigator in the source documents. 
The following minimum information should be recorded for each adverse reaction: 
- Adverse event description 
- Start and stop date of the adverse reaction 
- Severity 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 15 van 23 
 
 
 
- Seriousness 
- Causality assessment to the study interventions 
- Outcome 
 
6.2 Reporting to the Ethics Committee 
 
The sponsor will assess whether any relevant safety information that becomes available 
during the study should be reported ad hoc to the Ethics Committee of the University 
Hospitals Leuven, Belgium.  
The sponsor has the obligation to, once a year throughout the clinical trial (or on 
request), submit a progress report to the Ethics Committee of the University Hospitals 
Leuven, Belgium containing an overview of all serious adverse events that occurred 
during the reporting period and taking in to account all new available safety information 
received during the reporting period. 
 
7. Statistics 
 
7.1 Sample size 
Although this is a pilot study, we have calculated our sample size based on the data on 
SARS-CoV-2 positive Belgian citizens (58 517 011, data from Sciensano, bulletin of the 1st 
of May 2020) with a confidence interval of 5 and a confidence level of 95%. This will be 
the largest investigated cohort so far.  
7.2 Analysis 
 
The statistical analyses will be performed using statistical software such as R-studio or 
SPSS. The Shapiro-Wilk test will be used for normality testing. Depending their 
distribution, continuous variables will be summarized as mean ± standard deviation or 
median with interquartile range. Categorical variables will be summarized in absolute 
numbers and percentages. All statistical test will be significant if the P-value ≤ 0.05. 
Primary outcome measures 
The presence or absence of the virus in the tears is the primary objective. A multivariate 
analysis will be used to interpret the questionnaire.  
 
Secondary outcome measures 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 16 van 23 
 
 
 
For secondary outcome measures, we first have to indicate the different disease stages. 
We define them as early, intermediate and late stadium, respectively indicated by onset 
disease symptoms (cough, fever, fatigue, myalgia and lesser reported symptoms such ass 
headache, diarrhea and abdominal pain, ageusia and anosmia, conjunctivitis), 
development of chest pains and dyspnea with need for hospitalization and eventually 
development of SARS and need for ICU administration (12). We also define a remission 
stage in which symptoms start to decline.   
For investigation of the disease stage in which the SARS-nCoV-2 becomes detectable in 
the tears we aim to use an ANOVA analysis.  
8. Quality assurance 
 
To assure maximal quality and reproducibility, the trial protocol will be followed 
rigorously. 
9. Direct access to source data and documents 
 
Only data gathered in the context of the trial will be used. 
10. Ethics and regulatory approvals 
 
The trial will be conducted in compliance with the principles of the Declaration of 
Helsinki (2008), the principles of Good Clinical Practice and in accordance with all 
applicable regulatory requirements. This protocol and related documents will be 
submitted for review to Ethics Committee of the University Hospitals Leuven.  
 
The Study will be conducted only on the basis of prior informed consent by the Subjects 
to participate in the Study. We shall obtain a signed informed consent form (ICF) for all 
patients prior to their enrollment and participation in the Study in compliance with all 
applicable laws, regulations and the approval of the (local) Ethics Committee, if required. 
We shall retain such ICFs in accordance with the requirements of all applicable 
regulatory agencies and laws.  
11. Data Handling 
 
We shall treat all information and data as confidential and shall not disclose such 
information to any third parties or use such information for any purpose other than the 
performance of the Study. The collection, processing and disclosure of personal data, will 
comply with applicable personal data protection and the processing of personal data (the 
European General Data Protection Regulation of May 25, 2018). We will protect the data 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 17 van 23 
 
 
 
from disclosure outside the research according to the terms of the research protocol and 
the informed consent document. The subject’s name or other identifiers will be stored 
separately from their research data and replaced with a unique code to create a new 
identity for the subject. 
 
12. Data Management 
 
The questionnaires will be collected by Websurveyserver KU Leuven and all data will be 
stored in on a secured database named Redcap, which is protected within the firewall of 
the University Hospitals Leuven, Belgium with a unique pseudonymised identifier for 
every subject. In the future, these data could also be used for related projects. 
 
13. Publication Policy 
 
Any publication will be submitted to all co-authors at least thirty (30) days prior to 
submission or disclosure. 
 
14. Financial Aspects 
 
No commercial conflict of interest is related to this study. There will be no financial 
compensation for the volunteers.  
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 18 van 23 
 
 
 
15. References  
 
1.  
Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia 
in China, 2019. N Engl J Med. 2020 Feb;382(8):727–33.  
2.  
World Health Organization. Coronavirus disease 2019 ( COVID-19 ) Situation report - 51 [Internet]. 
2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-
reports 
3.  
Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel 
coronavirus in Wuhan, China. Lancet (London, England). 2020 Feb;395(10223):497–506.  
4.  
Vaira LA, Salzano G, Deiana G, De Riu G. Anosmia and ageusia: common findings in COVID-19 patients. 
Laryngoscope. 2020 Apr;  
5.  
Johnlee Varghese. New COVID 19 symptom: Eye pain too could be one of Coronavirus symptoms, says 
data scientist based on Google Trends [Internet]. 7 April. 2020. Available from: 
https://www.ibtimes.sg/new-covid-19-symptom-eye-pain-too-could-be-one-coronavirus-symptoms-
says-data-scientist-based-42522 
6.  
Wu P, Duan F, Luo C, Liu Q, Qu X, Liang L, et al. Characteristics of Ocular Findings of Patients With 
Coronavirus Disease 2019 (COVID-19) in Hubei Province, China. JAMA Ophthalmol. 2020 Mar;  
7.  
Xia J, Tong J, Liu M, Shen Y, Guo D. Evaluation of coronavirus in tears and conjunctival secretions of 
patients with SARS-CoV-2 infection. J Med Virol. 2020 Feb;  
8.  
Loon S-C, Teoh SCB, Oon LLE, Se-Thoe S-Y, Ling A-E, Leo Y-S, et al. The severe acute respiratory 
syndrome coronavirus in tears. Br J Ophthalmol. 2004 Jul;88(7):861–3.  
9.  
Moreira LB, Sanchez D, Trousdale MD, Stevenson D, Yarber F, McDonnell PJ. Aerosolization of 
infectious virus by excimer laser. Am J Ophthalmol. 1997 Mar;123(3):297–302.  
10.  
Hagen KB, Kettering JD, Aprecio RM, Beltran F, Maloney RK. Lack of virus transmission by the excimer 
laser plume. Am J Ophthalmol. 1997 Aug;124(2):206–11.  
11.  
Britt JM, Clifton BC, Barnebey HS, Mills RP. Microaerosol formation in noncontact “air-puff” tonometry. 
Arch Ophthalmol (Chicago, Ill  1960). 1991 Feb;109(2):225–8.  
12.  
Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel 
coronavirus in Wuhan, China. Lancet (London, England). 2020 Feb;395(10223):497–506.  
 
 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 19 van 23 
 
 
 
16. Addenda 
 
Addendum A: Questionnaire to be filled in by all subjects 
 
BIJKOMSTIGE VRAGENLIJST EYE-COVID STUDIE 
 
 
Deelnemerscode:  
 
Geboortedatum:   DD/MM/JJJJ 
 
Geslacht:   Man/Vrouw 
 
1ste dag ontstaan symptomen: DD/MM/JJJJ 
 
 
 
Geef aan of u volgende symptomen al dan niet ervaren heeft. Indien u ‘Ja’ antwoordde op het ervaren 
van symptomen, gelieve dan in de uiterst rechtse kolom de datum aan te geven waarop u deze 
symptomen voor het eerst heeft opgemerkt.  
 
 
Vraag 
Aanwezigheid symptomen? 
 
 
 
Datum ontstaan 
symptomen 
(DD/MM/JJJJ) 
Hebt u een verandering in de waarneming van geuren 
opgemerkt de afgelopen dagen?  
Ja  
Nee 
 
…../…../…… 
Hebt u een verandering in smaak waargenomen de 
afgelopen dagen?  
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen roodheid aan één of beide ogen 
opgemerkt?  
Ja 
Nee 
 
…../…../…… 
Hebt u irritatie/pijn aan een of beide ogen ervaren in de 
voorgaande dagen? 
Ja 
Nee 
 
…../…../…… 
Hebt u de laatste dagen meer last gehad van tranende 
ogen?  
Ja 
Nee 
 
…../…../…… 
Hebt u etterige secreties thv de ogen gehad de 
voorgaande dagen? 
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen neusloop ervaren?  
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen een verstopte neus ervaren? 
Ja 
Nee 
 
…../…../…… 
Moest u sinds kort meer en/ of heviger hoesten?  
Ja 
Nee 
 
…../…../…… 
Bent u de afgelopen dagen kort van adem geweest? 
Ja 
Nee 
 
…../…../…… 
Hebt u pijn op de borst ervaren de voorgaande dagen? 
Ja 
Nee 
 
…../…../…… 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 20 van 23 
 
 
 
Hebt u de afgelopen dagen koorts gemeten (een 
temperatuur boven de 38,5°)? 
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen spierpijnen ervaren?  
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen hoofdpijn ervaren?  
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen last gehad van diarree? 
Ja 
Nee 
 
…../…../…… 
Hebt u de afgelopen dagen last gehad van 
buikpijnklachten? 
Ja 
Nee 
 
…../…../…… 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 21 van 23 
 
 
 
Addendum B: Explanation of the sampling 
 
1. Bij het afnemen van het staal draai je de borstel in de tegengestelde richting van de 
beweging. Voor het linker oog is dat tegenwijzerzin, voor het rechter oog is dat wijzerzin 
(geheugensteuntje: wrijven weg van de neus, draaien naar de neus)  
2. Plaats de borstel in het universeel transport medium (UTM) 
3. Plaats het UTM met staal in de koelkast op -18°C 
4. Binnen de 24 u moet het UTM met staal verplaatst worden naar -80°C  
 
 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 22 van 23 
 
 
 
 

 
 
Tear film SARS-nCoV-2 detection in symptomatic and pauci-symptomatic patients. 
Eye-COVID V8.0 dd 16-09-2020 
 
 
 
 
Pagina 23 van 23 
 
 
 
 
Addendum C: Poster/flyer for the recruitment