Detection of SARS-CoV-2 nucleic acid in CSF by ultrahigh depth sequencing in a patient with COVID-19 and neurological dysfunction: a case report

Background: SARS-Coronavirus-2 (SARS-CoV-2), the pathogen of coronavirus disease 2019 (COVID-19), not only infects the respiratory tract, but also other organs. About a third of the inpatients of COVID-19 have neurological symptoms and in vitro experiments revealed that SARS-CoV-2 could infect human neural progenitor cells and brain organoids. However, the traditional test often reports negative owing to the low number of virus in the cerebrospinal uid. To date, timely diagnosis of central nervous system infection of SARS-CoV-2 remains a challenge. Case presentation: On day 14 of COVID-19, seizures, maxillofacial convulsions, intractable hiccups and signicant increase in intracranial pressure developed in a 56-year-old man. The RT-PCR of SARS-CoV-2 was negative. SARS-CoV-2 nucleic acid were detected in cerebrospinal uid (CSF) by ultrahigh depth sequencing. The patient was successfully treated after 14 days of mechanical ventilation and treatment of pneumonia and neurological dysfunction. Conclusions: This case suggests SARS-CoV-2 can invade the central nervous system and relevant examinations with CSF including depth sequencing among COVID-19 patients neurological


Background
Coronavirus disease 2019 (COVID-19) is a newly illness and has become a pandemic threat leading to more than 30 million con rmed cases and more than 950,000 deaths globally, as of September 18th, 2020. SARS-Coronavirus-2 (SARS-CoV-2), the pathogen of COVID-19, predominantly involves the lungs and causes respiratory illness [1]. However, this virus not only infects the respiratory tract, but also other organs, including intestinal tract, urinary system, blood and so on [2]. Furthermore, this virus has been also reported to be associated with meningitis/encephalitis [3]. In a study of hospitalized COVID-19 patients in Wuhan, China, 36.4% of patients were found to have neurological symptoms including headache, anosmia, ageusia, confusion, seizure, and encephalopathy [4]. Recently, a study reported that SARS-CoV-2 can productively infect human neural progenitor cells and brain organoids, highlighting the potential of direct viral involvement in neurological symptoms in COVID-19 patients [5]. However, proof of a direct involvement of SARS-CoV-2 is missing in the most cases of COVID-19 with central nervous system (CNS) symptoms have been reported until now, because the virus or nucleic acid was not detected in cerebrospinal uid (CSF) [6][7][8]. Here, we report a case of involvement of the CNS by the SARS-CoV-2, which was con rmed by ultrahigh depth metagenomic next generation sequencing (mNGS).

Case Presentation
On 24 January 2020, a 56-year-old man was admitted to the hospital due to fatigue, dizziness for 7 days, and fever for 3 days (Fig. 1). The patient had hypertension and a history of travel to Wuhan 14 days prior to hospitalization and one relative was diagnosed with COVID-19 two days ago. Computed tomography (CT) scan of the chest revealed a large area of ground-glass opacities (GGO) dominated by extraneous areas in both lungs (Fig. 2), and throat swab SARS-CoV-2 nucleic acid test by real-time reverse transcription-polymerase chain reaction (RT-PCR) was positive. Then COVID-19 with respiratory failure was con rmed in this patient. A nasal catheter was inserted and was oxygenated at 5 L/min. He was given antiviral therapy with lopinavir/ritonavir (500 mg twice daily) combined with interferon alfa-2b (5 million units twice daily, atomisation inhalation), moxi oxacin (0.4g once daily, intravenously) to prevent secondary infection (Fig. 1). After admission, the symptoms of dyspnea kept worsening. On day 10 of illness, chest CT showed an enlarged GGO area and partial opacities in both lungs. Short-term high-ow nasal oxygen was brie y administered with a gas ow rate of 50 L/min and oxygen concentration of 90%. The patient continued to exhibit respiratory distress accompanied with RR 50 times/min, SpO 2 85%.
Therefore, endotracheal intubation was performed in the intensive care unit (ICU) and mechanical ventilation was conducted according to the respiratory ventilation protocol of severe acute respiratory distress syndrome.
After 96 hours of mechanical ventilation, frequent maxillofacial and oral spasms accompanied by persistent hiccoughs were observed during the reduction of midazolam and fentanyl citrate. Physical examination revealed positive neck-resistance, bilateral pupils of equal size (3 mm diameter) with slow response to light, increased muscle tension in the extremities, hyperre exia in both knees, and positive bilateral Babinski sign and ankle clonus. The CSF pressure was greater than 330 mmH 2 O with a clear colourless appearance. Three days later CSF test showed that the pressure was greater than 290 mmH 2 O, the CSF cell count was 5/mL, the protein was 30mg/mL and glucose was 4.3mmol/L (Fig. 1), and no abnormalities were found in the brain CT (Fig. 3).
Total genomic DNA and RNA was extracted from the CSF for RT-PCR and mNGS to identify potential pathogens. The RT-PCR of SARS-CoV-2 was negative. Ultrahigh depth mNGS was done and, the full data set of 209,119,576 raw reads was obtained from the RNA library, which gave 10,116 sequences that showed 99.99% identity and covered 100% of the SARS-CoV-2 genome NC_045512.2|SARS-CoV-2|Wuhan-Hu-1 (GenBank accession no. NC_045512.2) with the average sequencing depth was 31.6 (Supplementary gure 1). Except for the SARS-CoV-2, no other pathogens were detected. Furthermore, assembled 29,003 bp and 15,790 bp SARS-CoV-2 genomes were obtained from sputum and blood samples respectively based on 61,224,674 and 8,800,232 raw reads, respectively. No SNP was found among the three assembled SARS-CoV-2 sequences from CSF, sputum and blood. Sequence comparison with the reference genome of Wuhan-Hu-1 revealed only three amino acid residue mutations dispersed in S2 subunit of spike protein, ORF3 and ORF8 (Supplementary gure 2). This CSF derived genome did not form special branch in the phylogenetic tree generated from the comparison of the epidemic virus genomes obtained from GenBank (Supplementary gure 3).
After 14 days of mechanical ventilation and treatment of pneumonia and neurological dysfunction, pulmonary lesions gradually improved, and neurological symptoms disappeared. The endotracheal intubation was removed on day 24 of illness, and the patient was discharged from ICU on day 32 of illness. The head MR was examined on May 6 (day 82 of illness) and high signal shadows were found in the hippocampus and bilateral temporal lobe, which may be the affected lesion (Figure 1). The patient had no cognitive barriers and memory impairment after discharge.

Discussion And Conclusions
Human coronaviruses are recognized as respiratory viruses. However, among these recognized human respiratory pathogens, at least HCoV-OC43, HCoV-229E, and SARS-CoV could be associated with the triggering or the exacerbation of neurological diseases as viral RNA or infectious virus can be detected in human brains [9][10][11]. Preliminary reports showed some COVID-19 patients showed CNS manifestations, such as dizziness, headache, nausea, vomiting, impaired consciousness, acute cerebrovascular disease, ataxia, and seizure, which warned that SARS-CoV-2 could have neuroinvasive potential [4][5][6][7][8][9][10][11][12][13]. Similar to SARS-CoV, SARS-CoV-2 also bind to the angiotensin-converting enzyme 2 (ACE2) receptor to enter human cells [14]. Many types of cells in the brain, such as neurons and glial cells, expressing ACE2 and may act as targets and are thus vulnerable to SARS-CoV-2 infection, and in vitro experiments revealed that SARS-CoV-2 could infect human neural progenitor cells and brain organoids [5]. In our patients, the main neurological symptoms are maxillofacial convulsion, intractable burping, intracranial pressure signi cantly increased combined with neck resistance, positive bilateral Babinski sign and ankle clonus, which suggesting the presence of neurological dysfunction. Although our case does not con rm that these CNS symptoms are caused by the SARS-CoV-2, we can con rmed that SARS-CoV-2 had invaded the CNS, as whole genome of SARS-CoV-2 was obtained from CSF by mNGS.
In summary, although a direct association between the symptoms of encephalopathy and SARS-CoV

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of The Beijing Ditan Hospital, Capital Medical University. The written consent to publish this information has been obtained from the study patient.

Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author (HZ) upon reasonable request.

Figure 1
Timeline of disease course according to days from initial presentation of illness and days from hospital admission.

Figure 2
CT scan of the chest. Both lungs show scattered and patchy akes of ground-glass opacities (GGO) on 24 January (day 7 of illness).