In a recent study posted to the medRxiv* preprint server, researchers investigated the clinical, histopathological, imaging, and serological characteristics of post-acute sequelae of coronavirus disease 2019 (COVID-19) (PASC) in patients with mild COVID-19.

Background

Long coronavirus disease (COVID) or PASC is a persistent, multi-organ system syndrome reported by most patients who recovered from severe COVID-19 requiring hospitalization. Patients complain of long-lasting symptoms such as dyspnea, headaches, fatigue, musculoskeletal pain, cognitive impairments such as difficulty concentrating, anosmia, dysgeusia, and cardiovascular and gastrointestinal problem occurring from three months to a year after recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.

Studies have shown that even mild COVID-19 cases not requiring hospitalization have a 4–15% burden of PASC, lasting up to eight months after recovering from the SARS-CoV-2 infection. However, the pathophysiology of PASC in mild COVID-19 cases remains unclear, with contradicting or inconclusive results from various case studies on the clinical, serological, and histopathological features and imaging abnormalities associated with mild SARS-CoV-2 infections not requiring hospital or intensive care unit admissions.

About the study

The present study examined a cohort of unvaccinated patients with mild SARS-CoV-2 infections confirmed through positive reverse transcription polymerase chain reaction (RT-PCR) tests. A detailed patient history of allergies and previous medications was recorded, and a baseline diagnosis consisting of spiroergometry and a physical examination was conducted. All patients were also screened for antinuclear antibody (ANA),  antineutrophil cytoplasmic antibody (ANCA), and extractable nuclear antigen (ENA).

Diagnostic imaging comprised a dual-energy computed tomography (DECT) scanner to acquire a multi-slice, non-contrast, spiral CT of the lungs, which was then used to determine the lung volume, overinflation, or attenuation of the lung with evidence of fibrosis. Additionally, transbronchial biopsies were conducted to obtain lung tissue samples, which then underwent histological staining. The tissue samples were observed for signs of post-viral change, such as fibrosis, peribronchiolitis, alveolitis, goblet cell hyperplasia, smooth muscle hypertrophy, and squamous epithelial metaplasia in the airways.

Additionally, fluorescence in situ hybridization (FISH) and immunohistochemistry were performed to detect the SARS-CoV-2 envelope gene, and spike and nucleocapsid proteins, respectively, in the lung tissue samples. The lung tissues were also observed under an electron microscope. Immunofluorescence multiplex staining was performed on the lung tissue samples for T lymphocyte subtyping to detect cells positive for cluster of differentiation (CD)16, CD68, CD16/CD163, and CD68/CD163, as well as other cell types. The expression of S100 calcium-binding protein A9 (S100A9) was also measured. Furthermore, enzyme-linked immunosorbent assay (ELISA) was performed using LEGENDplex immunoassay to detect and analyze cytokines in bronchoalveolar lavage samples.

Results

The results reported that the prevalent pulmonary symptom in the cohort was dyspnea during exertion, with the maximal expiratory flow at 50% (MEF50) and forced expiratory volume in 1 second (FEV1) being less than 80% in 42.9% and 16% of the patients, respectively. Additionally, airflow obstruction was observed in 35.3% of the patients with low attenuation volume in more than 5% of the lung area. Autoantibodies were detected in 33.3% of the patients, but the results did not correlate with the imaging or histopathological results, or the severity of symptoms.

Histopathological characterization revealed fibrin deposition in the alveoli and interstitial and peribronchiolar lymphocytosis in the lung tissue. Lung tissue from PASC patients also showed evidence of organizing pneumonia. However, no nucleocapsid or spike proteins were detected in the lung tissue, and the envelope gene was detected only in the lung tissue of one patient.

The macrophage phenotyping revealed a greater number of CD4⁺ T cells than CD8⁺ T cells in the small airways and alveoli. The inflammation and number of CD4⁺ T cells were greater in the small airways than in the interstitium. The electron microscopy images showed fibrosis and collagen fibril deposition in the interstitial region. Although the population of pro-fibrotic CD68⁺CD163⁺ S100A9 macrophage phenotype was higher in the lung tissues of PASC patients compared to those of the control cohort, the difference was not significant. Additionally, elevated levels of interleukin (IL)-1β and IL-8 in the bronchoalveolar lavage fluid were detected during the immunoassay.

Conclusions

Overall, the results suggested that although increased low attenuation volume and lower MEF50 and FEV1, along with evidence of fibrosis in the alveoli and bronchiolitis with predominant CD4⁺ T cells was found in PASC patients, the inflammation was limited to the small airways, and no detectable viral reservoirs were observed. Therefore, whether the pulmonary symptoms of PASC, such as dyspnea in patients with mild cases of COVID-19, are caused specifically by SARS-CoV-2 or are a general response to viral infections remains unclear.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.