cornea doi: 10.1097/ico.0000000000002479...10 bartz-schmidt, md (1); jens martin rohrbach, md (1);...
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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
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
1. Keil SD, Ragan I, Yonemura S, et al. Inactivation of severe acute respiratory 331
syndrome coronavirus 2 in plasma and platelet products using a riboflavin and 332
ultraviolet light-based photochemical treatment. Vox Sang. 2020 Apr 20. doi: 333
10.1111/vox.12937. [Epub ahead of print] 334
2. CDC COVID-19 Response Team. Characteristics of Health Care Personnel with 335
COVID-19 - United States, February 12-April 9, 2020. MMWR Morb Mortal Wkly 336
Rep. 2020 Apr 17;69(15):477-481. doi: 10.15585/mmwr.mm6915e6. 337
3. Judson TJ, Odisho AY, Neinstein AB, et al. Rapid Design and Implementation of 338
an Integrated Patient Self-Triage and Self-Scheduling Tool for COVID-19. J Am Med 339
Inform Assoc. 2020 Apr 8. pii: ocaa051. doi: 10.1093/jamia/ocaa051. [Epub ahead of 340
print] 341
4. Hu K, Patel J, Patel BC. Ophthalmic Manifestations Of Coronavirus (COVID-19). 342
StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020-. 343
2020 Apr 13. 344
5. Chen L, Liu M, Zhang Z, et al. Ocular manifestations of a hospitalised patient with 345
confirmed 2019 novel coronavirus disease. Br J Ophthalmol. 2020 Apr 7. pii: 346
bjophthalmol-2020-316304. doi: 10.1136/bjophthalmol-2020-316304. [Epub ahead of 347
print] 348
6. Barton LM, Duval EJ, Stroberg E, et al. COVID-19 Autopsies, Oklahoma, USA. 349
Am J Clin Pathol. 2020 Apr 10. pii: aqaa062. doi: 10.1093/ajcp/aqaa062. [Epub 350
ahead of print] 351
Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
14
7. Centers for Disease Control and Prevention. Collection and Submission of 352
Postmortem Specimens from Deceased Persons with Known or Suspected COVID-353
19. (2020) Available from: https://www.cdc.gov/coronavirus/2019-354
ncov/hcp/guidance-postmortem-specimens.html 355
8. Food and Drug Administration. Updated Information for Human Cell, Tissue, or 356
Cellular or Tissue-based Product (HCT/P) Establishments Regarding the 357
Coronavirus Disease 2019 Pandemic (April 1, 2020). Available from: 358
https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/updated-359
information-human-cell-tissue-or-cellular-or-tissue-based-product-hctp-360
establishments 361
362
9. Gérard D, Henry S, Thomas B. SARS-CoV-2: A new aetiology for atypical 363
lymphocytes. Br J Haematol. 2020 Apr 20. doi: 10.1111/bjh.16730. [Epub ahead of 364
print] 365
10. Di Mascio D, Khalil A, Saccone G, et al. Outcome of Coronavirus spectrum 366
infections (SARS, MERS, COVID 1 -19) during pregnancy: a systematic review and 367
meta-analysis. Am J Obstet Gynecol MFM. 2020 Mar 25:100107. doi: 368
10.1016/j.ajogmf.2020.100107. [Epub ahead of print] 369
11. Xia J, Tong J, Liu M, et al. Evaluation of coronavirus in tears and conjunctival 370
secretions of patients with SARS-CoV-2 infection. J Med Virol. 2020 Feb 26. doi: 371
10.1002/jmv.25725. [Epub ahead of print] 372
12. Peng Y, Zhou YH. Is novel coronavirus disease (COVID-19) transmitted through 373
conjunctiva? J Med Virol. 2020 Mar 16. doi: 10.1002/jmv.25753. [Epub ahead of 374
print] 375
Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
15
13. Liu Z, Sun CB. Conjunctiva is not a preferred gateway of entry for SARS-CoV-2 376
to infect respiratory tract. J Med Virol. 2020 Apr 10. doi: 10.1002/jmv.25859. [Epub 377
ahead of print] 378
14. Irene C Kuo. (2020) A Rashomon Moment? Ocular Involvement and COVID-19. 379
Ophthalmology, S0161-6420(20)30404-8. doi: 10.1016/j.ophtha.2020.04.027. Online 380
ahead of print. 381
15. Walls AC, Park YJ, Tortorici MA, et al. Structure, Function, and Antigenicity of 382
the SARS-CoV-2 Spike Glycoprotein. Cell. 2020 Apr 16;181(2):281-292.e6. doi: 383
10.1016/j.cell.2020.02.058. Epub 2020 Mar 9. 384
16. Senanayake Pd, Drazba J, Shadrach K, et al. Angiotensin II and its receptor 385
subtypes in the human retina. Invest Ophthalmol Vis Sci. 2007 Jul;48(7):3301-11. 386
17. Wagner J, Jan Danser AH, Derkx FH, et al. Demonstration of renin mRNA, 387
angiotensinogen mRNA, and angiotensin converting enzyme mRNA expression in 388
the human eye: evidence for an intraocular renin-angiotensin system. Br J 389
Ophthalmol. 1996 Feb;80(2):159-63. 390
18. Demurtas P, Di Girolamo N, Corrias M, et al. Immunohistochemical analysis of 391
angiotensin converting enzyme in Sardinian pterygium. Histol Histopathol. 2013 392
Jun;28(6):759-66. doi: 10.14670/HH-28.759. Epub 2012 Dec 4. 393
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
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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