Study population and design

Patients who were diagnosed with COVID-19 infection and admitted to the emergency department of Peking Union Medical College Hospital from December 2022 to January 2023 participated in this prospective study. All of the patients signed informed consent forms and the study was approved by the hospital’s ethics committee. This study was approved by the Ethics Committee of PUMCH and registered at chictr.org.cn (identifier ChiCTR2000030349). Inclusion criteria were (1) duration of hospitalization ≥ 48 h and (2) confirmed severe and critical COVID-19 infections. The exclusion criteria were (1) mild and moderate patients with confirmed COVID-19 infection and (2) all patients who used antiviral drugs during their illness, including before the visit to the doctor. In accordance with the diagnosis and treatment protocol for COVID-19 (9th & 10th edition) established by the National Health Commission of the People’s Republic of China, COVID-19 infection is defined as having (1) clinical manifestations related to COVID-19 infection and (2) one or more of the following etiological and serological test results: (a) positive COVID-19 nucleic acid test, (b) positive COVID-19 antigen, (c) positive COVID-19 isolation and culture, and (d) a level of novel coronavirus-specific immunoglobulin G antibody in the convalescent phase that is equal or greater than four times that in the acute phase. The clinical classification was defined as follows. “Mild” referred to the mild manifestations of the main clinical symptoms of upper respiratory tract infection, such as dry throat, sore throat, cough, and fever. “Moderate” referred to persistent high fever for > 3 days or cough, or both, shortness of breath with a respiratory rate (RR) < 30 times/min and oxygen saturation > 93% when breathing air at rest, and imaging that showed the characteristic manifestations of COVID-19 pneumonia. A classification of “severe” was used for adults who met any of the following criteria that could not be explained by anything other than COVID-19 infection: (a) shortness of breath (i.e., RR ≥ 30 times/min), (b) oxygen saturation < 93% when breathing air at rest, (c) arterial partial oxygen pressure/oxygen absorption concentration ≤ 300 mmHg (1 mm Hg = 0.133 kPa), and (d) progressively worsening clinical symptoms and lung imaging that showed that the lesion had progressed significantly (i.e., > 50%) within 24 to 48 h. Critical patients were defined as those who has one of the following conditions occurs: (1) respiratory failure requiring mechanical ventilation; (2) shock; (3) complicated with other organ failure requiring ICU care.

All of the enrolled patients were labeled as being in early (0–7 days), convalescent (8–14 days), and advanced (> 14 days) stages based on their symptoms (fever, sore throat, cough, and dyspnea) or the time of their first positive COVID-19 nucleic acid test. Since the criteria for glucocorticoids were severe and critical patients with progressive deterioration of oxygenation indexes, rapid imaging progress, and excessive activation of the body’s inflammatory response according to the diagnosis and treatment protocol for COVID-19 (9th edition) established by the National Health Commission of the People’s Republic of China, after enrollment, at least two experienced clinicians assessed the need for glucocorticoids, which were administered orally at a dose of 5 mg dexamethasone daily.

Clinical and laboratory evaluation

All of the enrolled patients underwent a comprehensive clinical and laboratory evaluation on the day of hospitalization. The information recorded included age, sex, underlying diseases, important biochemical indicators of infection (e.g., procalcitonin, creatinine, albumin, bilirubin, and coagulation), Acute Physiology And Chronic Health Evaluation II (Apache-II) [14] and Sequential Organ Failure Assessment (SOFA) [15] scores, and a record of any life-sustaining treatments that lasted ≥ 24 h (e.g., mechanical ventilation, non-invasive positive pressure ventilation, high-flow nasal catheter, Venturi mask and extracorporeal membrane oxygenation, vasopressor drugs, or renal replacement therapy).

Immunological laboratory examination

Serum was obtained from all of the patients to test immune parameters, including white blood cell count, complement, immunoglobulin, inflammatory factors, T cell subsets, and ferritin. In brief, the detection of T cell subsets began with the isolation of peripheral blood mononuclear cells, which were then stained with different combinations of fluorescent monoclonal antibodies. Finally, T cells (CD3+), CD4+ T cell subsets (CD4+CD3+ and CD28+CD4+), CD8+ T cell subsets (CD8+CD3+, CD28+CD8+), B cells (CD19+), and NK cells (CD3−CD16+CD56+) were detected using flow cytometry.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) detection of COVID-19 viral load in throat swabs

Throat swabs were taken daily from all of the enrolled patients for COVID-19 viral load testing. The patients were instructed to rinse their mouths with water and a swab was inserted in the mouth and passed over the base of the tongue. Both sides of the pharyngeal tonsils were first swabbed back and forth at least three times and then we’re swabbed over and down the back wall of the pharynx at least three times. The swab was withdrawn and placed into a collection tube. All of the swabs were collected and soaked in 1,000 μl phosphate buffer saline. After 30 s of shaking, 400 μl of the sample was removed for nucleic acid extraction with the SY619 nucleic acid extraction kit (Suzhou Xinbo Biotechnology Co. Ltd., Suzhou, China) in accordance with the manufacturer’s instructions. The number of viral copies was measured using RT-PCR using primers and probes that target the N gene of the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). The reference standard was diluted tenfold from 1 × 108 copies to 1 × 109 copies. The PCR amplification cycle was 15 min at 50 °C, 3 min at 95 °C, 15 s at 95 °C, 45 s at 60 °C and then plate-read for 50 cycles. The amplification process, fluorescence signal detection, data storage, and analysis were all completed by quantitative fluorescence PCR and its software Bio-Rad CFX Manager (Bio-Rad Laboratories Co. Ltd., California, USA). The number of copies of the virus was calculated according to the standard curve and then converted to log 10 for statistical analysis. COVID-19 negative conversion was defined as two consecutive cycle threshold (CT) values of SARS-CoV-2 nucleic acid N gene and ORF gene ≥ 35.

Statistical analysis

All analyses were performed using SPSS for Windows version 24.0 (IBM Corp., Armonk, NY, USA). The Kolmogorov–Smirnov test, Student’s t-test, and Mann–Whitney U test were used to examine the cumulative distribution functions and analyze the normally distributed continuous and nonparametric variables. The chi-square or Fisher exact test was chosen as appropriate for the comparison of categorical variables. P values associated with “equal variances not assumed” were reported for variables that violated the homogeneity of variance assumption. All tests performed were two-tailed, and P < 0.05 was considered statistically significant.