Case report: Persistence of residual antigen and RNA of the SARS-CoV-2 virus in tissues of two patients with long COVID

Abstract

The World Health Organization has defined long COVID-19 (LC) as a condition that occurs in individuals with a history of SARS-CoV-2 infection who exhibit persistent symptoms after its acute phase that last for at least two months and cannot be explained by an alternative diagnosis. Since we had previously reported residual viral antigens in tissues of convalescent patients, we aimed to assess the presence of such antigens in long COVID tissues. Here, we established the presence of the residual virus in the appendix, skin, and breast tissues of 2 patients who exhibited LC symptoms 163 and 426 days after symptom onset. With multiplex immunohistochemistry, we detected viral nucleocapsid protein in all three tissues. The nucleocapsid protein was further observed to colocalize with macrophage marker CD68, suggesting that immune cells were direct targets of SARS-CoV-2. Additionally, using RNAscope, the presence of viral RNA was also detected. Our positive finding in the breast tissue is corroborated by the recent reports of immunocompromised patients experiencing LC symptoms and persistent viral replication. Overall, our findings and emerging LC studies raise the possibility that the gastrointestinal tract may function as a reservoir for SARS-CoV-2.

Keywords: long covid; multiplex immunohistochemistry; post-acute COVID-19 syndrome; residual SARS-CoV-2; viral persistence.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Goh, Lim, Fernaíndez, Joseph, Edwards, Neo, Lee, Caballero, Lau and Yeong.

Figures

Figure 1

Residual SARS-CoV-2 detected in the appendix and skin tissues of patient 1 using multiplex immunohistochemistry and RNAscope (A, D) Representative images of the (A) appendix and (D) skin tissues stained with hematoxylin and eosin, with differentiated staining of nuclear (hematoxylin) and cytoplasmic (eosin) components. (B, E) Representative images of the (B) appendix and (E) skin tissues stained for DAPI (blue), CK/EpCAM (red), CD45 (cyan), SARS-CoV-2 nucleocapsid protein (COVID NP; green), and CD68 (yellow). (C, F) Representative images of the (C) appendix and (F) skin tissues stained for DAPI (blue), SARS-CoV-2 nucleocapsid protein (COVID NP; green), and CD68 (red). (G–I) Representative images of the (G, H) appendix tissue obtained from patient 1 and (I) normal colon tissue obtained from patients not affected by COVID-19, subjected to RNAscope in situ hybridization with a nuclear component (hematoxylin) counterstained. SARS-CoV-2 spike RNAs are labelled as green dots, examples of the green dots are marked by black arrows. (A–F) Scale bar, 100 μm. (G–I) Scale bar, 20 μm.

Figure 2

Residual SARS-CoV-2 detected in the breast tissue of patient 2 using multiplex immunohistochemistry and RNAscope (A) Representative images of the breast tissue stained with hematoxylin and eosin, with differentiated staining of nuclear (hematoxylin) and cytoplasmic (eosin) components. (B) Representative images of the breast tissue stained for DAPI (blue), CK/EpCAM (red), CD45 (cyan), SARS-CoV-2 nucleocapsid protein (COVID NP; green), and CD68 (yellow). (C) Representative images of the breast tissue stained for DAPI (blue), SARS-CoV-2 nucleocapsid protein (COVID NP; green), and CD68 (red). (D–F) Representative images of (D, E) the breast tissue obtained from patient 2 and (F) normal breast tissue obtained from patients not affected by COVID-19, subjected to RNAscope in situ hybridization with a nuclear component (hematoxylin) counterstained. SARS-CoV-2 spike RNAs are labelled as green dots, examples of the green dots are marked by black arrows. (A–C) Scale bar, 100 μm. (D–F) Scale bar, 20 μm.