Olfactory bulb SARS‐CoV‐2 infection is not paralleled by the presence of virus in other central nervous system areas

The ongoing SARS-CoV-2 pandemic has proven to be challenging, in both clinical and pathological terms. The viral tropism for angiotensin-converting enzyme 2 (ACE-2) receptor-expressing cells is paralleled in the clinical setting by the development of a spectrum of manifestations, most notably acute respiratory distress syndrome (ARDS). 1 – 3 Neurological symptoms are frequent in COVID-19 patients; however, central nervous system (CNS) infection and subsequent neurological disease attributable to SARS-CoV-2 remains a still poorly defined topic. 4,5 Both glial and neuronal cells have demonstrated ACE-2 expression and are therefore susceptible to SARS-CoV-2 infection. 6 – 8 Moreover, the possible spread of the virus through olfactory nerve fibres to the CNS remains an interesting although elusive issue. 9 A number of post-mortem neuropathological studies have reported vascular, thrombotic, and ischaemic alterations in COVID-19 cases. 10 – 16 Of note, the presence of SARS-CoV-2 within CNS specimens was reported to range between 0% and 53% of analysed cases across studies, including the olfactory bulbs and/or

The ongoing SARS-CoV-2 pandemic has proven to be challenging, in both clinical and pathological terms. The viral tropism for angiotensinconverting enzyme 2 (ACE-2) receptor-expressing cells is paralleled in the clinical setting by the development of a spectrum of manifestations, most notably acute respiratory distress syndrome (ARDS). [1][2][3] Neurological symptoms are frequent in COVID-19 patients; however, central nervous system (CNS) infection and subsequent neurological disease attributable to SARS-CoV-2 remains a still poorly defined topic. 4,5 Both glial and neuronal cells have demonstrated ACE-2 expression and are therefore susceptible to SARS-CoV-2 infection. [6][7][8] Moreover, the possible spread of the virus through olfactory nerve fibres to the CNS remains an interesting although elusive issue. 9 A number of post-mortem neuropathological studies have reported vascular, thrombotic, and ischaemic alterations in COVID-19 cases. [10][11][12][13][14][15][16] Of note, the presence of SARS-CoV-2 within CNS specimens was reported to range between 0% and 53% of analysed cases across studies, including the olfactory bulbs and/or cerebral parenchyma. [12][13][14][15][16] Our group aimed to describe the neuropathological findings in SARS-CoV-2 infected patients who died during the COVID-19 pandemic and, by using real-time (RT) PCR and immunohistochemistry The entire brain was fixed in neutral-buffered formalin for a minimum of 21 days (range 21-42 days). Macroscopic examination and sampling were performed by cutting the brain in the coronal plane at level of the mammillary bodies and then sectioning at 1 cm intervals in order to comprehensively evaluate brain parenchyma and related structures. An extensive sampling of the brain was performed, which included areas from cervical spinal cord, brain stem (medulla oblongata, pons and midbrain), cerebellar hemispheres (cortex and dentate nuclei), cerebral hemispheres (frontal, parietal, temporal, and occipital cortex; hippocampus with entorhinal cortex, basal ganglia, thalamus, hypothalamus, amygdala, olfactory tubercles with medial and lateral olfactory tracts), and olfactory bulbs. Other organs, that is, lungs, heart, liver, kidneys, spleen, and bowel, were extensively sampled for diagnostic purposes and were routinely examined.
For histopathological examination, 3 μm thick sections from each brain paraffin block were stained with haematoxylin and eosin. IHC was performed on the most representative areas in cases with signs of inflammation, with antibodies to glial fibrillary acidic protein (GFAP-clone EP672Y), macrophages (CD68-clone KP1), T-lymphocytes (CD3-clone 2GV6), B-lymphocytes (CD20-clone L26), and granulocytes (myeloperoxidase-polyclonal antibody). All antibodies were ready-to-use (Ventana, Roche Diagnostics, Basel,  Our results are summarised in Table 1

CONFLICT OF INTEREST
The authors declare no conflict of interests.

ETHICS STATEMENT
The Ethics Committee of Luigi Sacco Hospital approved the use of patient data for scientific research related to the disease. This study followed the Italian general rules used for scientific research purposes (regulation no. 72-26/03/2012).

AUTHOR CONTRIBUTIONS
GL was responsible for the original draft preparation, data collection and curation, and final draft preparation; CT, GO, EL, MB, and GC were responsible for the data collection and curation; LC, AP, and RSR performed autopsies; PZ performed autopsies and was responsible for the data collection and curation; MN performed autopsies and was responsible for the supervision, data collection and curation, and final draft preparation. All authors approved the final version of the manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.