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1

CORNEA 1

DOI: 10.1097/ICO.0000000000002479 2 3

TITLE PAGE 4

5

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in Human 6

Corneal Tissues 7

8

Tarek Bayyoud, MD(1, *); Angelika Iftner (2); Thomas Iftner, PhD (2); Karl Ulrich 9

Bartz-Schmidt, MD (1); Jens Martin Rohrbach, MD (1); Marius Ueffing, PhD (1, 3); 10

Michael Schindler, PhD (2); and Sebastian Thaler, MD 11

12

Department of Ophthalmology (1) University Hospital Tübingen, Tübingen, Germany, 13

(2) Institute for Medical Virology, University Hospital Tübingen, Tübingen, Germany 14

and (3) Institute for Ophthalmic Research (3), University of Tübingen, Tübingen, 15

Germany 16

17

*Corresponding author's complete contact information: Department of 18

Ophthalmology, University Hospital Tübingen, Tübingen, Germany, Elfriede-Aulhorn-19

Str. 7, 72076 Tübingen, Tarek Bayyoud, MD, Telephone: +49-7071-29-88088, Email: 20

tarek.bayyoud@med.uni-tuebingen.de 21

22

23

24

No conflict of interest or any funding to disclose. 25

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2

Keywords: SARS-CoV-2; COVID-19; corneal transplantation; tissue procurement 26

and processing 27

TITLE: Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in 28

Human Corneal Tissues 29

Tarek Bayyoud, MD; Angelika Iftner; Thomas Iftner, PhD; Karl Ulrich Bartz-Schmidt, 30

MD; Jens Martin Rohrbach, MD; Marius Ueffing, PhD; Michael Schindler, PhD, and 31

Sebastian Thaler, MD 32

PURPOSE: To examine corneal tissue for SARS-CoV-2 positivity with regard to 33

implications for tissue procurement, processing, corneal transplantation and ocular 34

surgery on healthy patients. We performed quantitative (q)RT-PCR-testing for 35

SARS-CoV-2 RNA on corneal stroma and endothelium, bulbar conjunctiva, 36

conjunctival fluid swabs, anterior chamber fluid and corneal epithelium of COVID-19 37

postmortem donors. 38

METHODS: Included in this study were 10 bulbi of 5 COVID-19 patients who passed 39

away due to respiratory insufficiency. Informed consent and Institutional Review 40

Board approval was obtained prior to this study (241/2020BO2). SARS-CoV-2 was 41

detected via a pharyngeal swab and broncho-alveolar lavage. Tissue procurement 42

and tissue preparation were performed with personal protective equipment (PPE) 43

and the necessary protective measures. qRT-PCR-testing was performed for each of 44

the above mentioned tissues and intraocular fluids. 45

RESULTS: The qRT-PCRs yielded no viral RNA in the following ocular tissues and 46

intraocular fluid: Corneal stroma and endothelium, bulbar-limbal conjunctiva, 47

conjunctival fluid swabs, anterior chamber fluid and corneal epithelium. 48

CONCLUSION: In this study no SARS-CoV-2-RNA was detected in conjunctiva, 49

anterior chamber fluid and corneal tissues (endothelium, stroma and epithelium) of 50

COVID-19 donors. This implicates that the risk for SARS-CoV-2 infection via corneal 51

or conjunctival tissue is very low. However, further studies on a higher number of 52

COVID-19 patients are necessary to confirm these results. This might be of high 53

importance for donor tissue procurement, processing and corneal transplantation. 54

55

56

57

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INTRODUCTION: 58

The ongoing Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) 59

pandemic is a global health threat that causes immanent hardships in medical 60

practice involving logistics, patient management, surgery and handling of infectious 61

materials1-3.One of the first healthcare professionals to raise the alarm was the 62

Chinese ophthalmologist Li Wenliang, who died from coronavirus disease 2019 63

(COVID-19) at the young age of 33, after being infected by one of his patients4. In 64

general, ophthalmologists are at great risk due to close patient contact while 65

performing a significant number of examinations and interventions. Despite the 66

question, whether SARS-CoV-2 has the potential to be transmitted via ocular fluids, 67

it is of special interest for cornea and eye banking specialists to know if corneal 68

tissues are potentially infectious and possibly mediate the transmission of SARS-69

CoV-2 from corneal donors to recipients. Currently there is a lot of discussion among 70

corneal specialists regarding acute adjustments and changes to standard 71

procedures in tissue procurement, processing and transplantation (personal 72

communication, Tissue transplant Section of the German Ophthalmological Society). 73

A recent study confirmed that SARS-CoV-2 can invade the conjunctival epithelium 74

and cause a full-blown picture of viral conjunctivitis5. The objective of this study was 75

to evaluate corneal involvement in COVID-19 postmortem donors in the following 76

tissues: corneal stroma and endothelium, bulbar-limbal conjunctiva, conjunctival fluid 77

swabs, anterior chamber fluid and corneal epithelium. Findings may have 78

implications for corneal transplantation and in particular corneal tissue procurement 79

and processing. Our secondary objective was to describe precautions taken and 80

personal protective equipment (PPE) used during these tissue procurements6. 81

82

83

MATERIALS AND METHODS: 84

INFORMED CONSENT, APPROVAL OF INDEPENDENT INSTITUTIONAL 85

REVIEW BOARD 86

Informed consent, adherence to the Declaration of Helsinki and approval of an 87

independent Ethics Committee (institutional review board) was obtained prior to 88

commencement of study (241/2020BO2). 89

90

TISSUE PROCUREMENT 91

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Specific guidelines, assessment of the environment for tissue procurement, 92

and personal protective equipment: 93

Guidelines for the enucleation team (two persons): To be checked prior to the 94

enucleation of a COVID-19 postmortem donor: 95

1. Place of enucleation defined as an area and/or room needing permission to 96

access (time spent at location has to be documented); 97

2. Place of enucleation is not allowed to be used by another person at time of tissue 98

extraction; 99

3. Any kind of aerosol and/ or turbulence has to be prevented; 100

4. The necessary equipment has to be discarded after usage and/ or disinfected 101

depending on the specific utensils used; 102

5. To preclude any kind of self-harm personal protective equipment (PPE) has to be 103

used appropriately. This includes: 104

surgical hand disinfection; 105

gowns (overalls and apron); double gloves (as indicator system); and hood; 106

face mask (FFP-3 level: 0.6µm/ 99% filtration); 107

surgical instruments with tray; 108

Disposal of infectious wastes in a one-time lockable container; and of sharp 109

utensils in a suitable, second container. 110

111

Enucleation and preparation protocols: 112

A routine tissue procurement protocol for corneal banking was employed for the left 113

globe of each donor. The respective right globe was kept naïve during the 114

enucleation and preparation steps. 115

116

117

Enucleation: 118

The enucleation was performed at the designated COVID-19 autopsy room of the 119

Institute for Pathology and Neuro-pathology of the University Hospital of Tuebingen. 120

The average time of death to retrieval was 21 hours. 121

The following steps were performed: 122

Final check of the set of instruments; 123

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Double check identity of postmortem donor and consent form; 124

Confirm cause of death (COVID-19); 125

Documentation of donor side (right/left), place, and time of enucleation; 126

Proper usage of PPE including fitting test of FFP-3/ N-95; 127

Inspection of body bag and corpus; 128

Preparation of transport media and vessel (left globe: sterile gauze, 10mL 129

NaCl, 10 mL gentamicinsulfate (5mg/mL); right globe: sterile gauze, 10mL 130

NaCl; mark each vessel: COVID-19 donor tissue); 131

Flushing of the superior and inferior fornix of the left globe (Betaisodona; 132

1:10 diluted in sterile NaCl equivalent to 1% of free iodine, flushing with sterile 133

NaCl after 5 min), periocular wiping with Betaisodona; right globe is kept 134

naive; 135

Appropriate usage of provided drape, vessel and PPE; 136

Perform enucleation with provided single-use surgical set (eyelid blocker, 137

forceps, scissors, hooks) to obtain an intact globe with conjunctivae (5-138

10mm); 139

Prosthesis selection, insertion and closure of palpebral fissures; 140

Transfer of each globe into specific transport vessel and a re-lockable 141

container marked “COVID-19 donor tissue”; 142

Disposal of used PPE and potentially infectious materials. 143

Transport: 144

Transport via re-lockable, marked container (“COVID-19 donor tissue”); 145

Temperature is recorded and kept between 33.8°F to 50°F (+1 to +10°C) 146

using cooling packs and box avoiding contact to ice (Libero T1, Elpro, 147

Switzerland); direct preparation and further testing of donor tissue or storage 148

at 42.8°F (6°C); 149

Preparation: 150

The preparation was performed at a BSL2 laboratory (under a sterile workbench) of 151

the Institute for Medical Virology of the University of Tuebingen. The average time of 152

death to preservation was 31 hours. The following steps were performed: 153

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Use of PPE including fitting test of FFP-3/ N-95; 154

Disinfection of the sterile workbench (Descosept-AF, desiccation of 15 min); 155

Disinfection of globe (left) in diluted iodine solution (5 min in Betaisadona® 156

(7.5 %, Braun, #3864154)/ NaCl (250 ml, Fresenius Kabi, PZN-00809049) 157

1:20) and thorough rinse (in 50 mL NaCl); right globe is kept naïve; 158

Preparation of a corneoscleral donor tissues/fluids (surgical set: surgical 159

forceps,15 mm trephine, 30G-cannulas, Kolibri-forceps, Vannas-scissors, 160

Westcott-scissors, hockey knife and vacuum holder) and extraction of tissue 161

samples for quantitative Reverse Transcription-Polymerase Chain Reaction 162

(qRT-PCR) testing on SARS-CoV-2 RNA (4 samples per type of tissue/fluid: 163

corneal stroma and endothelium, bulbar conjunctiva, conjunctival fluid swabs, 164

anterior chamber fluid and corneal epithelium); 165

RNA extraction and quantitative Reverse Transcription-Polymerase Chain 166 Reaction based on quality approved protocols with controls: 167

Addition of 600µL RLT (1mL RLT, 10µL ß-mercapto-ethanol RNeasy Kit, 168

QiaCube, QiaSymphony DSP Virus/ Pathogen Kit, Qiagen, Hilden, Germany;) 169

and one 5mm-steel ball (Qiagen #69989) to each sample; 170

Dissolution in ball mill (Fa. Retsch, 2min, level 100); 171

Purification in shredder pillar (Qiagen (#79656), centrifugation at 2min at 172

14.000rpm; addition of same volume of 70% EtOH with DEPC-H2O , non-173

vortex mix, 700µL for RNeasy spin column, centrifugation at 15sec at 174

14.000rpm, repeat with remaining RLT/EtOH mix); 175

qRT-PCR using RealStar SARS-CoV-2 RT-PCR Kit 1.0 (altona Diagnostics 176

GmbH, Hamburg, Germany) and LightMix® Modular SARS-CoV (COVID19) 177

kit (TIB Molbiol Syntheselabor GmbH, Berlin, Germany). 178

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179

DNAse digest with RNAse-Free DNase Set (#79254), purification with RNeasy 180

Mini Kit (Qiagen #74106): 181

Dissolution of lyophilized DNAse in RNAse free water (550µL); 182

Addition of RW1 buffer ( centrifugation at 15sec at 14.000rpm, wash 183

column) and of DNAse (10µL) to RDD buffer (70µL), non-vortex mix, 184

centrifugation; 185

Addition of DNAse mix to center of column (15min RT); 186

Add RW1 buffer (350µL) to column ( centrifugation at 15sec at 14.000rpm), 187

RPE (500µL, centrifugation at 15sec at 14.000rpm, wash column); 188

Transfer column to 2nd collection tube ( centrifugation at 1min at 14.000rpm) 189

and new Eppendorf tube/ micro-reaction vessel; 190

Add RNAse free water (30-50µL, centrifugation at 1min at 14.000rpm); 191

Second addition of RNAse free water (30-50µL, centrifugation at 1min at 192

14.000rpm) in case of >30µg RNA. 193

quantitative Reverse Transcription-Polymerase Chain Reaction: 194

10 µl of the RNA, positive or negative control were used for qRT-PCR with the 195

LightMix® SarbecoV E-gene Kit (TIB MOLBIOL, 40-0776-96) in combination 196

with the Roche LightCycler® Multiplex RNA Virus Master (Roche, 197

07083173001). The positive Control was supplied with the LightMix Kit and 198

contained all diagnostic targets (E gene, N gene and RdRP) of SARS and 199

SARS-CoV-2. As negative control the water supplied with the Roche Master 200

kit was used. The reaction mix was prepared as described in the manual. 201

Data analysis was performed as described in LightCycler II operator’s manual, 202

in brief, color compensation was selected for multiplex assays and the 203

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“Second Derivative Maximum method” was used. The results were shown in 204

FAM channel. 205

According to the producers manual, the sensitivity is 5.2 copies per reaction. 206

A hole genome, synthetic RNA control (Twist Bioscience, #MT007544.1) was 207

also used in qRT-PCR; a consecutive dilution showed, that down to 10 copies 208

per reaction SARS-CoV-2 was detectable (linear correlation) (data not 209

shown). The cut-off was defined as recommended in the LightMix Kit manual: 210

CP value for 10 copies (35.48 ± 0.2) plus 1 cycle and resulted in a CP-cut-off 211

value of 36.48. 212

Postmortem pulmonary tissue samples from COVID-19 deceased were tested for 213

SARS-CoV-2 RNA via RT-PCR. All tested samples had positive SARS-CoV-2 results 214

(unpublished data). Whether these samples were still infectious or not was not 215

evaluated. 216

The interim guidance of the Centers for Disease Control and Prevention states 217

concerning "Collection and Submission of Postmortem Specimens from Deceased 218

Persons with Known or Suspected COVID-19": No data are currently available on 219

the frequency of detection of SARS-CoV-2, the virus that causes COVID-19, by RT-220

PCR on postmortem swabs collected at different durations after death. If COVID-19 221

testing on postmortem swab specimens is being considered for a suspected COVID-222

19 case, SARS-CoV-2 RNA may still be detected up to 3 days postmortem and 223

possibly longer based on available data from experiences with MERS-CoV and 224

SARS-CoV; however sensitivity may be reduced with a longer postmortem interval, 225

and duration of illness may need to be considered in interpreting a negative result7. 226

Per the United States Food and Drug Administration, respiratory viruses, in general, 227

are not known to be transmitted by transplantation of human cell, tissue, or cellular 228

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9

or tissue‐based product and there have been no reported cases of SARS‐CoV, 229

MERS‐CoV, or any other coronavirus transmission via transplantation of ocular 230

tissue8. European agencies advance a similar view as the US-centric agencies. This 231

is outlined in the European CDC Technical Report “Infection prevention and control 232

for COVID-19 in healthcare settings – first update, 12 March 2020”. It references the 233

“World Health Organization Interim Guidance for Collection and Submission of 234

Postmortem Specimens from Deceased Persons Under Investigation (PUI) for 235

COVID-19, 19 February 2020” (cited 11 March 2020; available from: 236

https://www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-postmortem-237

specimens.html). 238

Histopathology: 239

A histopathological macroscopic and microscopic examination using standard 240

hematoxylin and eosin stains was performed on the extracted tissues. 241

Donor characteristics and clinical aspects of COVID-19: 242

The age of the donors ranged from 74 to 89 (mean: 80 years; 1 female; 4 male). 243

Past medical history included: arterial hypertension in all and diabetes mellitus in one 244

patient. All donors were on anti-hypertensive drug regimens. One patient received in 245

addition anti-hyperglycemic treatment. Three patients were on angiotensin-246

converting enzyme (ACE) inhibitor class of medications, one patient was on an 247

angiotensin-receptor blocker (ARB) and another one on the anti-hyperglycemic 248

agent of the biguanide class (metformin). The mean time of hospitalization prior to 249

demise was 15 days (±12.9 SD; range: 1-32 days)Tab1a. 250

All patients had initially unspecific symptoms which progressed to a full picture of 251

COVID-19 with distinct dyspnea. Pharyngeal swabs and bronchoalveolar lavage fluid 252

were tested positive for SARS-CoV-2-RNA by qRT-PCR. Coinfection by HSV, CMV, 253

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RSV, parainfluenza, and influenza were excluded through qRT-PCR. The type of 254

care included supportive, respiratory intubation and machine-assisted support 255

including extracorporeal membrane oxygenation (ECMO) and continuous 256

venovenous hemofiltrationTab1b. Supportive care was administered to all patients, 257

respiratory ventilation to 4 patients and machine-assisted support to 3 patients (1x 258

ECMO and 2x hemofiltration). Organ system involvement was extensive in all cases 259

and included the respiratory, gastrointestinal and urogenital systems. The respiratory 260

system was involved in all cases extending to Acute Respiratory Distress Syndrome 261

in all and complicated by pleural effusion in two, atrial fibrillation in one and 262

myocardial infarction in three patients. A life-threatening organ dysfunction was 263

diagnosed in all patients leading to the involvement of the gastrointestinal and 264

genitourinary systems. Acute liver failure was seen in two and acute kidney failure in 265

all patients. Multi-organ dysfunction syndrome was finally diagnosed in 3 patients. 266

Laboratory parameters showed a leukocytosis combined with lymphopenia in 2 and 267

a reduced hemoglobin concentration in 4 cases. 268

RESULTS: 269 We report here the absence of SARS-CoV-2 RNA in corneal tissues obtained from 270

COVID-19 postmortem donors using qRT-PCR. All tissue samples tested negative 271

for SARS-CoV-2 viral RNA amplyfing the viral S and E genesTab2. All internal positive 272

and negative controls were valid and included in each set of analyses. In addition, 273

there was no difference noted in SARS-CoV-2 RNA detection between the routine 274

tissue procurement protocol for corneal banking employed for the left globe of each 275

donor and the respective right globe which was kept naïve during the enucleation 276

and preparation steps. 277

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The macroscopic and microscopic histopathological examinations performed 278

confirmed in all globes normal extra- and intraocular morphology without histological 279

signs of inflammation. 280

DISCUSSION: 281

Recent studies suggest that clinical manifestations of ocular surface disease of 282

COVID-19 are not common and are usually limited to the conjunctiva5,11-13. To our 283

knowledge our study may be the first suggesting the absence of SARS-CoV-2 RNA 284

in conjunctiva and corneal tissue in COVID-19 cadaveric donors. Recently, a case of 285

viral conjunctivitis of SARS-CoV-2 has been reported5. In addition, its RNA has been 286

detected in tears and conjunctival secretions11. This suggests that the clinical 287

spectrum of an ocular SARS-CoV-2 involvement might potentially be of greater 288

extent. 289

Related to the current Centers for Disease Control and Prevention interim guidance 290

we would like to point out that false negative testing may be due to timing of PCR 291

testing, testing capability, postmortem interval and length of hospitalization/ duration 292

of disease (see also [14]). In addition, no test has been validated to date for testing 293

in cadaveric donors. We note here that qRT-PCR was done on COVID-19 cadaveric 294

donor tissues and fluids. Thus, a false negative result might be more likely than 295

during the acute phase of the disease. However, the number of eligible cases was 296

limited because of informed consent of the next of kin or the patient himself prior to 297

demise. 298

Therefore, future independent studies analyzing higher numbers of postmortem 299

COVID-19 donors for SARS-CoV-2 RNA in ocular tissues are necessary and 300

warranted. Furthermore, to clarify possible modes of transmission through 301

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conjunctiva or ocular tissues evidence of viral replication and cytopathology in living 302

subjects suffering from COVID-19 should be analyzed in all phases of disease. 303

To our current knowledge, SARS-CoV-2 viral replication as well as its lytic activity 304

restricts to epithelia. Therefore, corneal epithelial cells could potentially host the virus 305

and when transplanted within corneal transplants may transmit virus to the recipients 306

of these transplants. This motivated us to analyze different types of corneal tissues 307

and anatomically related fluids of COVID-19 tissue donors for presence of SARS-308

CoV-2 RNA. 309

So far, little is known on the clinical spectrum of ocular disease caused by SARS-310

CoV-2 infection9,10. However, several modes of transmission of SARS-CoV-2 311

involving ocular tissue and tears are being discussed11-13. The angiotensin-312

converting enzyme 2 (ACE-2) receptor has been found to be a binding site of SARS-313

CoV-215. Separately, the presence of ACE-2 receptor has been noted in ocular 314

tissues15-18. 315

Due to these uncertainties regarding a possible transmission, we have adapted our 316

tissue procurement process for the collection of COVID-19 positive patients 317

according to the current guidelines and described it in detail in the context of this 318

study. Even though the increased safety precautions mean an increased expenditure 319

of time and material, we recommend taking them into account. No SARS-CoV-2 320

infection occurred in our collection team during this study. 321

In conclusion, this study shows the absence of SARS-CoV-2 RNA in postmortem 322

COVID-19 donors in corneal tissues and anatomically related fluids. This implicates 323

that the risk for SARS-CoV-2 infection via corneal or conjunctival tissue may be very 324

low and suggests that SARS-CoV-2 transmission via ocular tissue may be an 325

unlikely event. Taking into account the limitations of this study, it suggests a low risk 326

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for viral transmission due to tissue procurement and processing of donor tissue for 327

corneal transplantation surgery from individuals succumbed to SARS-CoV-2. 328

329

REFERENCES: 330

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394

ACKNOWLEDGEMENT: 395

This study was supported by the Institute of Pathology and Neuropathology, 396

Department of General and Molecular Pathology and Pathological Anatomy of the 397

University Hospital Tübingen. We thank Prof. Dr. Falko Fend and PD Dr. Hans 398

Bösmüller for their outstanding cooperation. 399

400

401

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402

COVID-19: Coronavirus disease 2019 403

ID: Identification number 404

d: Days 405

PMH: Past Medical History 406

aHTN: Arterial hypertension 407

DM: Diabetes mellitus 408

PDH: Past Drug History 409

aHT: Anti-hypertensive treatment 410

HCT: Hydrochlorothiazide 411

AGT: Anti-glycemic treatment 412

413 Tab. 1b COVID-19 patients: Type of care and organ system involvement

Pat. ID

Type of care Organ system involvement

Supportive Intubation ECMO Vv- Hemofiltration

ARDS Sepsis Liver Failure

Kidney Failure

MODS

1 yes yes yes yes yes yes yes yes yes

2 yes yes no yes yes yes yes yes yes

3 yes no no no yes yes no yes yes

4 yes yes no yes yes yes no yes no

5 yes yes no no yes yes no yes no

414

ECMO: Extracorporeal membrane oxygenation 415

Vv: venovenous 416

ARDS: Acute Respiratory Distress Syndrome 417

MODS: Multiple Organ Dysfunction Syndrome 418

419 Tab. 2 COVID-19 postmortem donor tissues and SARS-CoV-2 qRT-PCR results

Type of ocular tissue/ fluid

qRT-PCR for SARS-CoV-2 RNA on right eye

RNA yields (mean, ±SD in ng/µL)

qRT-PCR for SARS-CoV-2 RNA on left eye*

RNA yields (mean, ±SD in ng/µL)

Conjunctival fluid swabs

vRNA undetectable 46.8±33.9 vRNA undetectable 51.4±29.3

Bulbar conjunctiva vRNA undetectable 13.9±7.8 vRNA undetectable 49.6±81.7

Corneal epithelium vRNA undetectable 52.4±45.4 vRNA undetectable 16.2±10.6

Corneal stroma and endothelium

vRNA undetectable 16.3±19.7 vRNA undetectable 19.5±14.0

Anterior chamber fluid vRNA undetectable 6.5±11.7 vRNA undetectable 7.1±12.1

420

Tab. 1a COVID-19 patient characteristics

Pat. ID Age Sex Time of hospitalization (d) PMH PDH

aHTN DM aHT AGT

1 75 male 9 yes no metoprolol n/a

2 78 male 8 yes yes HCT, bisoprolol,

lercanidipine, candesartan

metformin

3 87 female 1 yes no torasemide, ramipril

n/a

4 89 male 25 yes no ramipril, torasemide,

thiazide

n/a

5 74 male 32 yes no Ramipril, bisoprolol

n/a

Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

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*Received routine procedure of tissue procurement for corneal banking 421

SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2 422

qRT-PCR: quantitative Reverse Transcriptase-Polymerase Chain Reaction (S-/E-genes, positive/ internal controls) 423

vRNA: viral RNA 424

SD: Standard deviation 425

Total No. of COVID-19 postmortem donors: N = 5 426

427