Clinical, imaging, serological, and histopathological features of pulmonary post-acute sequelae after mild COVID-19 (PASC) (preprint)

Background: A significant proportion of patients experience prolonged pulmonary, cardiocirculatory or neuropsychiatric symptoms after Coronavirus disease 2019 (COVID-19), termed post-acute sequelae of COVID (PASC). Lung manifestations of PASC include cough, dyspnea on exertion and persistent radiologic abnormalities and have been linked to viral persistence, ongoing inflammation and immune dysregulation. So far, there is limited data on lung histopathology and tissue-based immune cell subtyping in PASC. Methods: 51 unvaccinated patients (median age, 40 years; 43% female) with a median of 17 weeks (range, 2-55 weeks) after mild SARS-CoV-2 infection (without hospitalization) underwent full clinical evaluation including high-resolution computed tomography (HR-CT) and transbronchial biopsy. We used RT-PCR/FISH and immunohistochemistry (nucleocapsid/spike/CD3/CD4/CD8) for residual SARS-CoV-2 detection and T lymphocyte subtyping, respectively. We assessed interstitial fibrosis and macrophage profiles by transmission electron microscopy (TEM) and immunofluorescence multiplex staining, while cytokine profiling in broncho-alveolar lavage (BAL) fluid was performed by legendplex immunoassay. Results: Dyspnea on exertion was the leading symptom of pulmonary PASC in our cohort. In 16% and 42.9% of patients, FEV1 and MEF50 were [≤]80% and 35.3% showed low attenuation volume (LAV) in >5% of lung area, in line with airflow obstruction. There was a significant correlation between oxygen pulse and time since COVID (p=0.009). Histopathologically, PASC manifested as organizing pneumonia (OP), fibrinous alveolitis and increased CD4+ T cell infiltrate predominantly around airways (bronchiolitis), while the residual virus components were detectable in only a single PASC patient (2%). T cell infiltrates around small airways were inversely correlated with time since COVID, however, this trend failed to reach statistical significance. We identified discrete interstitial fibrosis and a pro-fibrotic macrophage subtype (CD68/CD163/S100A9) as well as significantly elevated interleukin 1{beta} in BAL fluid from PASC patients (p=0.01), but H-scores for fibrotic macrophage population did not correlate with severity of clinical symptoms or T cell infiltration. Interpretation: We show decreased FEV1/MEF50 and increased LAV in line with obstructive lung disease due to CD4+ T cell-predominant bronchiolitis as well as evidence of pro-fibrotic signaling in a subset of unvaccinated PASC patients. Since our results point towards self-limiting inflammation of small airways without detectable viral reservoirs, it remains unclear whether pulmonary symptoms in PASC are SARS-CoV-2-specific or represent a general response to viral infection. Still, evidence of pro-fibrotic signaling should warrant clincal follow-up and further research into possible long-time fibrotic remodeling in PASC patients.

SARS-CoV-2 RNA screening in routine pathology specimens (preprint)

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients' routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess a) organ tropism in samples from COVID-19-positive patients, b) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and c) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in four samples from confirmed COVID-19 patients, in two gastric biopsies, one colon resection, and one pleural effusion specimen, while all other specimens, particularly from patients with mild COVID-19 disease course, were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.