The involvement of Central Nervous System and sequence variability of Severe Adult Respiratory Syndrome – Coronavirus-2 revealed in autopsy tissue samples: a case report.

Background: The case presented here illustrates that interdisciplinary teamwork can be essential for the understanding of the COVID-19 disease presentation and enlightening of the pathophysiology. Case presentation: A 60-years-old overweight woman without any comorbidities was found dead in her apartment after 14 days of home isolation due to suspicion on the Coronavirus disease 2019 (COVID-19). She had reported symptoms of tachycardia, fever, and increasing respiratory diculty one day before her death. Due to the Danish legal act on sudden deaths a forensic autopsy was performed including a thorough examination and biosampling. The results of the forensic autopsy displayed sever densied, almost airless, rm lungs, and an unspecic reactive minimal focal perivascular inammation consisting of macrophages of the brain tissue. The nal diagnosis, COVID-19 with involvement of the central nervous system was established by use of the RT-RNA analysis on cerebrospinal uid, as well as by serologic detection of the specic antibodies for SARS-CoV-2 in cerebrospinal uid and serum. The genetic analysis displayed a 2 % variation between SARS-CoV-2 isolates recovered from the tracheal sample, cerebrospinal uid, and tissues from both lungs. Conclusion: The combination of all available results revealed that the cause of death was COVID-19 with severe pulmonary disease and neuroinvasion, as well as renal affection resulting in hyponatremia. To our knowledge, it was not shown previously that neuroinvasion could be conrmed by the detection of specic antibodies for SARS-CoV-2 and SARS-CoV-2 specic RNA in cerebrospinal uid. This case supports hypotheses that SARS-CoV-2 may cause central nervous system infection. The genetic distinction between SARS-CoV-2 isolates was done by whole-genome sequencing, where the isolate recovered from the cerebrospinal uid was the most different.

death. Due to the Danish legal act on sudden deaths a forensic autopsy was performed including a thorough examination and biosampling. The results of the forensic autopsy displayed sever densi ed, almost airless, rm lungs, and an unspeci c reactive minimal focal perivascular in ammation consisting of macrophages of the brain tissue. The nal diagnosis, COVID-19 with involvement of the central nervous system was established by use of the RT-RNA analysis on cerebrospinal uid, as well as by serologic detection of the speci c antibodies for SARS-CoV-2 in cerebrospinal uid and serum. The genetic analysis displayed a 2 % variation between SARS-CoV-2 isolates recovered from the tracheal sample, cerebrospinal uid, and tissues from both lungs.
Conclusion: The combination of all available results revealed that the cause of death was COVID-19 with severe pulmonary disease and neuroinvasion, as well as renal affection resulting in hyponatremia. To our knowledge, it was not shown previously that neuroinvasion could be con rmed by the detection of speci c antibodies for SARS-CoV-2 and SARS-CoV-2 speci c RNA in cerebrospinal uid. This case supports hypotheses that SARS-CoV-2 may cause central nervous system infection. The genetic distinction between SARS-CoV-2 isolates was done by whole-genome sequencing, where the isolate recovered from the cerebrospinal uid was the most different.

Background Coronavirus disease 2019 (COVID-19) is an infection caused by Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2) which arose in Wuhan Hubei Province, China in late 2019 1 . SARS-CoV-2 is a relatively large single-stranded RNA virus, that is, closely related to SARS-CoV 1 . It has since spread and caused more than 483.217 deaths worldwide (https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html). By the middle of July 2020, a total of 611 cases of death were con rmed in Denmark by the Health Authorities with 4.7 % of deaths to a total number of con rmed infected cases (https://www.ssi.dk/sygdomme-beredskab-ogforskning/sygdomsovervaagning/c/covid19-overvaagning). The National Danish Register includes deaths recorded within 30 days of COVID-19 infection detected and the COVID-19 is not necessarily being the main cause of death.
An autopsy is a diagnostic tool to elucidate the pathogenesis of the COVID-19, and to discover SARS-CoV-2's pathogenic characteristics. In this case, a forensic autopsy was performed due to the Danish legal act on sudden death. The autopsy was performed according to ISO 17020 standards and included a whole body post-mortem computer tomography (PMCT) prior to the external and internal examination, the thorough biosampling, and the additional supplementary toxicological and histopathological tests, as well as extensive microbiological analyses, were performed. According to recently published autopsy studies, the primary cause of death in individuals who died of COVID-19 was a respiratory failure with exudative diffuse alveolar damage 3 . Additionally, it has been revealed that the leading causes of death in COVID-19-positive patients are bronchopneumonia, massive pulmonary embolism, and alveolar haemorrhage 3,4 .
Symptoms such as ataxia, seizures, as well as a loss of smell and taste have led to the hypothesis that the virus may be also neurotropic, possible through ACE2-receptors that are expressed in the brain 5 . It has also been suggested that neuroinvasion of SARS-CoV-2 may play a role in the respiratory failure of COVID-19 patients. Here we describe a woman with probable central nervous system (CNS) involvement due to COVID-19.

Case Presentation Patient information.
A 60-years-old overweight woman (Body Mass Index 28.5), without prior conditions, was found dead in her bed by her partner. The circumstantial information in the police report revealed that she had been in home-isolation for 14 days due to suspicion of COVID-19 infection. In the days preceding her death, she reported symptoms of high puls (around 100 beats per minute), fever, and increasing respiratory di culty to her family. The partner had been in contact with the deceased continually during the 14 days and neighbours had done the grocery shopping, which was left in front of her main door. During the last days, the decendent had been in telephone contact with her primary physician as well as the emergency medical service. She had an appointment with her primary physician, which was meant to take place the day after she was found dead. Clinical records were checked for pre-existing medical conditions and medications and none were reported.
Diagnostic assessment.
Post-mortem CT-scan (PMCT). The lungs were uniformly consolidated bilaterally with air entrapment (Figure 1 A and B). The abdominal organs and the brain were without signi cant changes, apart from unspeci c diffuse moderate edema of the brain, a calci ed isolated process related to the pericardium and subtle calci cation of the mitral valves.
Autopsy and histology.
The autopsy revealed heavily densi ed, almost airless, rm lungs with a total weight of 1460 grams without signs of purulent secretion. Pleura was without brin deposits, but the mucosa was hyperaemic. Tracheobronchial lymph nodes were enlarged. The brain was edematous weighing 1512 grams (normal weight 1250 grams +/-20 grams 8 ), with slight attening of the gyri, unremarkable arachnoid, gray and white matter, and without signs of in ammation. The kidneys showed signs of acute shock.
The histological examination was performed according to standard protocols 6,7  Tissue from the CNS comprised cerebrum with the motoric cortex, basal ganglia, hippocampus, pons, and cerebellum. The meninges were sparsely represented. The tissue showed an unspeci c reactive minimal focal perivascular in ammation consisting of macrophages. There was no edematous change, vasculitis, microthrombi, and no deposition of complement fractions.
Myocardial tissue was with unspeci c subtle edema. Tissue from the liver, spleen, lymph nodes, kidneys, bone marrow, and suprarenal glands showed relevant unspeci c hyperplastic changes and slight acute tubular necrosis in the kidneys.
Toxicology. Toxicologic analyses of blood and vitreous uid for medication, illicit drugs, alcohol, diabetesrelated analyses, and sodium as well as pottasium concentration were performed. The results showed a treatment-related concentration of paracetamol. The post-mortem electrolyte concentrations showed hyponatremia (103 mmol/L, de ned <134 mmol/L 9 ) while the potassium concentration was as expected in post-mortem material (21 mmol/L).
RNA-extraction. RNA extraction was performed in the ISO Certi ed routine PCR diagnostic reference laboratory using automated nucleic acid extraction (MagNA Pure 96, Roche) according to manufacture recommendations. The detection of SARS-CoV-2 was performed by real-time reverse transcription PCRbased assay as described previously 10 performed on post-mortem isolates. SARS-CoV-2 was identi ed in the CSF with cycle threshold (Ct) at 38.23, lung tissue from both left and right lung with Ct-values at 28.18 and 25.10, respectively, and in the tracheobronchial specimen with Ct 26.76. The blood specimen and the heart tissue specimen were negative for SARS-CoV-2.
Multiplex RT-PCR. Multiplex RT-PCR-based assay for seventeen different respiratory tract virus (in uenza type A and B, respiratory syncytial virus type A and B, metapneumovirus, parain uenzavirus type 1, 2, 3 and 4, adenovirus, rhinovirus, enterovirus, parechovirus and coronavirus OC43, 229E, NL63, HKU1), was performed. Tissue samples from the heart and both lungs, as well as tracheobronchial secretion, blood, and CSF were analysed by this assay. None of the viruses was detected in any of the samples.
Culturing. The culturing of the CSF showed the growth of Staphylococcus capitis. Besides, the growth of Bacillus cereus was identi ed of tissue from the right lung. Both of ndings were interpreted as either postmortem bacterial migration or contamination.
SARS-CoV-2 speci c antibodies testing. Two enzyme-linked immunosorbent assays (ELISA), iFlash 1800 Chemiluminescence Immunoassay Analyser (YHLO Biotech Co., Shenzhen, China) and the Wantai SARS-CoV-2 Total Antibody ELISA (Beijing Wantai Biological Pharmacy Enterprise, Beijing, China), were performed for investigation of the serological pro le of the patient according to the manufacturer's instructions and, as it was described earlier 11 . We examined serum and CSF with both methods. These tests are CE-marked, but not validated for the detection of antibodies in CSF.
Sequencing of SARS-CoV-2. Total RNA was converted to cDNA with Superscript IV First-Strand Synthesis Kit (Thermo) using 11 μl of extracted RNA and random hexamers according to the manufacturer's speci cations. PCR based ampli cation of the viral genome was performed according to the ARTIC protocol using two separate multiplex primer pools reactions of the version 3 release of the primer set plug-in installed for the import and mapping of Oxford Nanopore reads. Demultiplexed Reads were imported as Oxford Nanopore reads and paired Illumina reads respectively. Reads were trimmed against the V3 primer set (n = 218) to remove primer sequences from subsequent analysis. Oxford Nanopore reads and Illumina reads were mapped individually to the MT135044 SARS-CoV-2 reference sequence at CLCs default settings. To enhance the fraction of the genome sequenced and read-coverage (depth), Nanopore and Illumina mappings were merged into one for sequences from each of the 4 samples. This mapping had the following number of mapped reads, fraction (%) of the genome sequenced and average read coverages: Spinal: 101181, 98%, 294.12; Tracheal: 346914, 99%, 1041.55; L lung: 5044442, 99.9%, 14041.95; R lung: 4431907, 100%, 12191.83. Consensus sequences were generated from the merged read mappings both at any coverage and at a minimum coverage of 5 reads and inserting N as a symbol at sequence positions not covered su ciently. Sequence reads from the four different samples were individually aligned with the reference sequence and manually inspected and corrected in BioEdit 7.2.5 12 .
Only inter-sample variants observed in the MiSeq mapping covered by ≥5 reads and also observed in the Nanopore mapping were included in the consensus sequences. Non-resolved variant sites were marked with an N in the consensus sequence and excluded from further analysis. After this curation step, the fraction of the genome sequenced (in %) were: Spinal: 92.9%; Tracheal: 98.9%; L lung: 99.9%, and R lung: 100%.
Genotyping of SARS-CoV-2 samples. The clade designation system (https://nextstrain.org/ncov/global) was used by downloading the Nextstrain Metadata set on the 28 th of May 2020. From this Metadata list, 27 sequences, representing all major SARS-CoV-2 clades (A1a, A2, A2a, A3, A6, B, B1, B2, and B4; only clades represented by at least 10 sequences were included) were selected along with the Wuhan-Hu-1 sequence (NC_045512). These 28 reference sequences were aligned with four patient sequences (named after their anatomical origin: L Lung, R Lung, Tracheal, and Spinal) in Mafft Sequences from three of the samples (Spinal, Tracheal, and R lung) had a dominant SNP at three sites in the ORF1ab gene ( Table 1), two of which (R lung and Tracheal) were both mixed mutations. In sequences from the R lung, the SNP (T) was present in 66% of the reads while the G shared with the sequences from other samples was present in 33% of reads. Two minor variants, C and A constituted 0.01% and 0.06% of the reads and probably is most likely background seen at the high coverage observed at this site. The SNPs in the spinal and the Tracheal samples resulted in changes in the encoded amino acid ( Table 2). The majority of viral sequences in the spinal sample encoded a Glycine (G) instead of a Serine (S) in the 3-C proteinase gene and the majority of viral sequences in the tracheal sample encoded a V aline (V) instead of a Glycine (G) in the NSP11 encoding gene.

Discussion And Conclusions
The arrangement of all available results revealed that the cause of death was COVID-19 with severe pulmonary disease and neuroinvasion, as well as renal affection resulting in hyponatremia. A previous report found that 36.4% of COVID-19 patients had neurological manifestations and that the rate of neurological symptoms was higher in patients with severe infection 16 . However, in a recent report from Japan, a 24-year-old man was diagnosed with COVID-19 related meningitis/encephalitis, where speci c RNA-SARS-CoV-2 was detected in CSF, while it was not detected in the nasopharyngeal swabs 17 . In our case, speci c RNA-SARS-CoV-2 was detected in the tracheal sample, CSF, and tissue from both lungs. CNS-infection in relation to COVID-19 is further supported, by a report where the presence of the virus in neural and capillary endothelial cells, in the frontal lobe of a patient with COVID-19 infection and neurological symptoms, were documented by electron microscopy 18 . The microscopic examination of the brain revealed no signs of encephalitis, microthrombi, or in ammation, which is in keeping with Menter T et al. 3 , who, apart from unspeci c microscopic ndings, registered a low viral load in neurological tissue.
Phylogenetic analysis showed that all four samples belonged to the same A2a clade. Most European SARS-CoV-2-sequences on Nextstrain as per 28 th of May 2020 belong to this clade (3177 of 4082; 77.8%). In Denmark, this clade is the most dominant (206 of 208; 99.03%) (https://nextstrain.org/ncov/europe). The phylogenetic analysis showed that the spinal uid sample differed the most from the other three samples. The reason for this is unknown, but we hypothesize that this could be due to the selection of more t viruses and/or founder effects/bottlenecks when moving between compartments, as has been demonstrated previously for other viruses 19 .
When it comes to sequencing/PCR-results you always have to consider the possible error sources. Also for the spinal sample, 7.9% of the genome was not covered or the quality was too low for analysis, which might have lead to some mutations not being detected. However, since this is post-mortem material, which cannot be recovered once it is used, and since we used state of the art methods, this limitation cannot be further addressed with currently available methods.
In the above-described case, the patient had been ill for 14 days before her death and this timeline is supported by the nding of SARS-CoV-2 speci c antibodies. As has been found by others 20 , also the microscopic changes in the lung tissue, in this case, were consistent with the timeline with the representation of severe diffuse alveolar damage in the early phase, but also in an organizing and proliferative intermediary phase, indicating a disease process lasting around one to two weeks with a continuing severe acute component affecting both the pulmonary parenchyma, respiratory epithelium, and vasculature 20,21,3 .
Biochemical analysis, in this case, showed severe hyponatremia, which is suspected to be the result of several components: kidney affection with tubular necrosis and possibly increased water intake during the disease. The deceased was isolated in the weeks before her death and information regarding food and water intake in the last days is not available. It is concluded, that hyponatremia is a secondary result of a prolonged disease course.
To our knowledge, this is the rst report were both speci c antibodies for SARS-CoV-2 and SARS-CoV-2 speci c RNA were detected in CSF. However, the detection of speci c antibodies for SARS-CoV-2, in CSF is not validated and the clinical signi cance is unclear. To conclude, this case supports hypotheses that SARS-CoV-2 may cause central nervous system infection. The pathogenesis, incidence, and clinical impact, as well as the detection of speci c antibodies for SARS-CoV-2 in CSF, need to be investigated further.  Phylogenetic analysis of patient sequences. Phylogenetic tree rooted with the Wuhan-Hu-1 sequence (NC_045512) and GISAID sequences, shown for example EPI ISL 420347 (GISAID identi er) A2 (Nextstrain clade) and the four patient sequences (Spinal, Tracheal, R lung, and L lung).