Persistent SARS-CoV-2 infection in patients seemingly recovered from COVID-19

Abstract

SARS-CoV-2 infection is clinically heterogeneous, ranging from asymptomatic to deadly. A few patients with COVID-19 appear to recover from acute viral infection but nevertheless progress in their disease and eventually die, despite persistent negativity at molecular tests for SARS-CoV-2 RNA. Here, we performed post-mortem analyses in 27 consecutive patients who had apparently recovered from COVID-19 but had progressively worsened in their clinical conditions despite repeated viral negativity in nasopharyngeal swabs or bronchioalveolar lavage for 11-300 consecutive days (average: 105.5 days). Three of these patients remained PCR-negative for over 9 months. Post-mortem analysis revealed evidence of diffuse or focal interstitial pneumonia in 23/27 (81%) patients, accompanied by extensive fibrotic substitution in 13 cases (47%). Despite apparent virological remission, lung pathology was similar to that observed in acute COVID-19 individuals, including micro- and macro-vascular thrombosis (67% of cases), vasculitis (24%), squamous metaplasia of the respiratory epithelium (30%), frequent cytological abnormalities and syncytia (67%), and the presence of dysmorphic features in the bronchial cartilage (44%). Consistent with molecular test negativity, SARS-CoV-2 antigens were not detected in the respiratory epithelium. In contrast, antibodies against both spike and nucleocapsid revealed the frequent (70%) infection of bronchial cartilage chondrocytes and para-bronchial gland epithelial cells. In a few patients (19%), we also detected positivity in vascular pericytes and endothelial cells. Quantitative RT-PCR amplification in tissue lysates confirmed the presence of viral RNA. Together, these findings indicate that SARS-CoV-2 infection can persist significantly longer than suggested by standard PCR-negative tests, with specific infection of specific cell types in the lung. Whether these persistently infected cells also play a pathogenic role in long COVID remains to be addressed. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: COVID-19; SARS-CoV-2; cartilage; nucleocapsid; parabronchial glands; post-mortem analysis; spike; syncytia.

Figure 1

Lung pathology in former COVID‐19 patients. Haematoxylin and eosin (H&E) staining showing characteristic pathological features of previous COVID patients (patient's code is at the top of each panel). Scale bars in all panels: 100 μm. (A) Diffuse alveolar damage (DAD) and parenchymal fibrosis. The pictures, taken from three different patients, show massive alveolar inflammation with almost complete occlusion of the alveolar lumen; delamination of pneumocytes, which appear grossly damaged and dystrophic; endoalveolar fibrin deposition; and fibrosis of the inter‐alveolar septa, which is massive in patient 183.21. (B) Thrombosis of a medium‐ (left) and small‐ (right) sized artery. The thrombotic artery on the left is surrounded by hyalin fibrotic tissue and flanked by congested small vessels. Thrombi are in different stages of organisation. (C) Perivascular inflammatory cap in a small thrombotic vessel. (D) Squamous metaplasia (pseudosyncytia) of the respiratory epithelium. Massive defoliation of the metaplasic alveolar cells, only a few of which maintain a cylindrical ciliated appearance. Massive congestion of the surrounding small vessels. (E) Presence of syncytial cells in two patients. In the upper panel, the syncytium is in the context of a delaminated alveolar structure and in proximity to an arteriole with a thickened wall and exfoliated endothelium.

Figure 2

Histology of bronchial cartilage. Haematoxylin and eosin (H&E) staining is shown for five previous COVID‐19 patients and one normal control who died from non‐COVID pneumonia. In previous COVID‐19 patients, chondrocytes appear randomly clustered. Several cells show marked basophilic degeneration, with increased size, dysmorphic nuclear structure, and peripheral halos. In several instances, the chondrocytes have become anucleated. Scale bars: 100 μm.

Figure 3

Immunohistochemistry (IHC) for SARS‐CoV‐2 spike and nucleocapsid (N) antigens. (A) Evidence of SARS‐CoV‐2 infection in bronchial cartilage chondrocytes. For each of the patients (indicated at the top of each pair of pictures), low‐ and high‐magnification pictures are shown; the magnified area is indicated by a dashed box. Most of the positive chondrocytes (in dark brown) show dysmorphic features. Scale bars in all panels: 100 μm. (B) Same as in panel A for the area surrounding the bronchial structures. Several mucosal glands proximal to the bronchial cartilage were positive for both spike and N antigens.

Figure 4

Serial sections of cartilage (A) and the peri‐bronchial area (B) of two former COVID‐19 patients. Serial sections (from top to bottom, arrow) from the same paraffin block were stained with antibodies against spike, N, ACE2, and TMEM16F, as indicated on the left of each picture. Representative pictures of cartilage and peri‐bronchial glands (panels A and B, respectively) are shown. For each picture, low‐ and high‐magnification images are shown; the magnified area is indicated by a dashed box. In most cases, positivity for immunodetection by all four antibodies (brown cells) was in superimposable regions. Scale bars: 100 μm.