Viral loads, lymphocyte subsets and cytokines in asymptomatic, mildly and critical symptomatic patients with SARS-CoV-2 infection: a retrospective study.

BACKGROUND: Tens of million cases of coronavirus disease-2019 (COVID-19) have occurred globally. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. The aim of the present study is to investigate the laboratory characteristics of the viral load, lymphocyte subset and cytokines in asymptomatic individuals with SARS-CoV-2 infection in comparison with those in symptomatic patients with COVID-19. METHODS: From January 24, 2020, to April 11, 2020, 48 consecutive subjects were enrolled in this study. Viral loads were detected by RT-PCR from throat-swab, sputum and feces samples. Lymphocyte subset levels of CD3 + , CD4 + , and CD8 + T lymphocytes, B cells and NK cells were determined with biological microscope and flow cytometric analysis. Plasma cytokines (IL2, IL4, IL5, IL6, IL8, IL10, TNF-α, IFN-α and IFN-γ) were detected using flow cytometer. Analysis of variance (ANOVA), Chi-square or Fisher's exact test and Pearson's Correlation assay was used for all data. RESULTS: Asymptomatic (AS), mild symptoms (MS) and severe or critical cases (SCS) with COVID-19 were 11 (11/48, 22.9%), 26 (54.2%, 26/48) and 11 cases (11/48, 22.9%), respectively. The mean age of AS group (47.3 years) was lower than SCS group (63.5 years) (P < 0.05). Diabetes mellitus in AS, MS and SCS patients with COVID-19 were 0, 6 and 5 cases, respectively, and there was a significant difference between AS and SCS (P < 0.05). No statistical differences were found in the viral loads of SARS-CoV-2 between AS, MS and SCS groups on admission to hospital and during hospitalization. The concentration of CD 3 + T cells (P < 0.05), CD3 + CD4 + T cells (P < 0.05), CD3 + CD8 + T cells (P < 0.01), and B cells (P < 0.05) in SCS patients was lower than in AS and MS patients, while the level of IL-5 (P < 0.05), IL-6 (P < 0.05), IL-8 (P < 0.01) and IL-10 (P < 0.01), and TNF-α (P < 0.05) was higher. The age was negatively correlated with CD3 + T cells (P < 0.05), CD3 + CD4 + T cells (P < 0.05), and positively correlated with IL-2 (P < 0.001), IL-5 (P < 0.05), IL-6 (P < 0.05) IL-8 (P < 0.05), and IL-10 (P < 0.05). The viral loads were positively correlated with IL-2 (P < 0.001), IL-5 (P < 0.05), IL-6 (P < 0.05) IL-8 (P < 0.05) and IL-10 (P < 0.05), while negatively correlated with CD 3 + T cells (P < 0.05) and CD3 + CD4 + T cells (P < 0.05). CONCLUSIONS: The viral loads are similar between asymptomatic, mild and severe or critical patients with COVID-19. The severity of COVID-19 may be related to underlying diseases such as diabetes mellitus. Lymphocyte subset and plasma cytokine levels may be as the markers to distinguish severely degrees of disease, and asymptomatic patients may be as an important source of infection for the COVID-19.

Clinical and molecular characteristics of COVID-19 patients with persistent SARS-CoV-2 infection.

The characteristics of COVID-19 patients with persistent SARS-CoV-2 infection are not yet well described. Here, we compare the clinical and molecular features of patients with long duration of viral shedding (LDs) with those from patients with short duration patients (SDs), and healthy donors (HDs). We find that several cytokines and chemokines, such as interleukin (IL)-2, tumor necrosis factor (TNF) and lymphotoxin α (LT-α) are present at lower levels in LDs than SDs. Single-cell RNA sequencing shows that natural killer (NK) cells and CD14+ monocytes are reduced, while regulatory T cells are increased in LDs; moreover, T and NK cells in LDs are less activated than in SDs. Importantly, most cells in LDs show reduced expression of ribosomal protein (RP) genes and related pathways, with this inversed correlation between RP levels and infection duration further validated in 103 independent patients. Our results thus indicate that immunosuppression and low RP expression may be related to the persistence of the viral infection in COVID-19 patients.

Circulating cell-free DNA, peripheral lymphocyte subsets alterations and neutrophil lymphocyte ratio in assessment of COVID-19 severity.

Cell destruction results in plasma accumulation of cell-free DNA (cfDNA). Dynamic changes in circulating lymphocytes are features of COVID-19. We aimed to investigate if cfDNA level can serve in stratification of COVID-19 patients, and if cfDNA level is associated with alterations in lymphocyte subsets and neutrophil-to-lymphocyte ratio (NLR). This cross-sectional comparative study enrolled 64 SARS-CoV-2-positive patients. Patients were subdivided to severe and non-severe groups. Plasma cfDNA concentration was determined by real-time quantitative PCR. Lymphocyte subsets were assessed by flow cytometry. There was significant increase in cfDNA among severe cases when compared with non-severe cases. cfDNA showed positive correlation with NLR and inverse correlation with T cell percentage. cfDNA positively correlated with ferritin and C-reactive protein. The output data of performed ROC curves to differentiate severe from non-severe cases revealed that cfDNA at cut-off ≥17.31 ng/µl and AUC of 0.96 yielded (93%) sensitivity and (73%) specificity. In summary, excessive release of cfDNA can serve as sensitive COVID-19 severity predictor. There is an association between cfDNA up-regulation and NLR up-regulation and T cell percentage down-regulation. cfDNA level can be used in stratification and personalized monitoring strategies in COVID-19 patients.

Gender-associated difference following COVID-19 virus infection: Implications for thymosin alpha-1 therapy.

Gender influences clinical presentations, duration and severity of symptoms, and therapy outcome in coronavirus disease 2019 (COVID-19) infection. Whether the immune response to Tα1 treatment for SARS-CoV-2 differs between the sexes, and whether this difference explains the male susceptibility to COVID-19, is unclear. This study aimed to investigate the efficiency and safety of Tα1 treatment and provide a basis for practically identifying gender differences characteristics and features of COVID-19. One hundred twenty-seven patients had COVID-19 symptoms and tested COVID19-positive (female 42.52%) in Wuhan union hospital were enrolled for medication. They were randomly divided into groups Control and Tα1 intervention. Seventy-eight patients received a subcutaneous injection of 1.6 mg Tα1, based on supportive treatment for 15 days. The control group included untreated 49 COVID19 patients closely matched for gender and age and received regular supportive treatment. In this retrospective analysis, we found that COVID-19-infected males reported more symptoms than COVID-19-infected females. A high degree of gender differences-related variability was observed in CRP and PCT levels and the cell counts of many lymphocyte subpopulations in the COVID-19 patients after Tα1 intervention. Levels of CRP and IL-6 were higher in Tα1-treated male group than Tα1-treated female group, while the level of PCT was significantly lower in Tα1-treated male group. Gender differences may be a factor in sustaining COVID-19 immunity responded to Tα1, male and female show statistically significant differences in relevance to cytokine production associated with the development of a more significant number of symptoms. This leaves the question of identifying gender-specific risk factors to explain these differences.

CD4 and CD8 Lymphocyte Counts as Surrogate Early Markers for Progression in SARS-CoV-2 Pneumonia: A Prospective Study.

BACKGROUND: COVID-19 pathophysiology and the predictive factors involved are not fully understood, but lymphocytes dysregulation appears to play a role. This paper aims to evaluate lymphocyte subsets in the pathophysiology of COVID-19 and as predictive factors for severe disease. PATIENT AND METHODS: A prospective cohort study of patients with SARS-CoV-2 bilateral pneumonia recruited at hospital admission. Demographics, medical history, and data regarding SARS-CoV-2 infection were recorded. Patients systematically underwent complete laboratory tests, including parameters related to COVID-19 as well as lymphocyte subsets study at the time of admission. Severe disease criteria were established at admission, and patients were classified on remote follow-up according to disease evolution. Linear regression models were used to assess associations with disease evolution, and Receiver Operating Characteristic (ROC) and the corresponding Area Under the Curve (AUC) were used to evaluate predictive values. RESULTS: Patients with critical COVID-19 showed a decrease in CD3+CD4+ T cells count compared to non-critical (278 (485 IQR) vs. 545 (322 IQR)), a decrease in median CD4+/CD8+ ratio (1.7, (1.7 IQR) vs. 3.1 (2.4 IQR)), and a decrease in median CD4+MFI (21,820 (4491 IQR) vs. 26,259 (3256 IQR)), which persisted after adjustment. CD3+CD8+ T cells count had a high correlation with time to hospital discharge (PC = -0.700 (-0.931, -0.066)). ROC curves for predictive value showed lymphocyte subsets achieving the best performances, specifically CD3+CD4+ T cells (AUC = 0.756), CD4+/CD8+ ratio (AUC = 0.767), and CD4+MFI (AUC = 0.848). CONCLUSIONS: A predictive value and treatment considerations for lymphocyte subsets are suggested, especially for CD3CD4+ T cells. Lymphocyte subsets determination at hospital admission is recommended.

Long-term infection of SARS-CoV-2 changed the body's immune status.

The outbreak of SARS-CoV-2-associated pneumonia, a disease called COVID-19, has caused a pandemic worldwide. To investigate the immune responses after infection of SARS-CoV-2 in non-critical patients may help to better understand the disease progression. We collected 334 confirmed COVID-19 cases including 212 still in hospital with nucleic acid test positive on halfway for SARS-CoV-2 and 122 discharged from hospital, compared specific antibodies, immune cells, and cytokine changes between the hospitalized and discharged patients. The hospitalized patients had a longer illness time compared with discharged patients. Analysis of viral loads explained long-term or persistent infection of SARS-CoV-2, which existed with the median time of 18.5 days of the positive nucleic acid test. Serum analysis showed that the specific anti-N IgG antibody was positive in all detected patients after infection of two weeks. Neutrophils, Monocytes, NK cells, and CD4+ T cells significantly increased, while total lymphocytes and CD8+ T cells decreased from non-critical hospitalized patients after longer-term infection. Further analysis of the cytokines showed that IL-6, TNF-α, IFN-γ, IL-2, IL-4, and IL-10 from the hospitalized patients were significantly higher, indicating a potential of the increased CD4+ T cell differentiation.