Free-Breathing Low-Field MRI of the Lungs Detects Functional Alterations Associated With Persistent Symptoms After COVID-19 Infection.

OBJECTIVES: With the COVID-19 pandemic, repetitive lung examinations have become necessary to follow-up symptoms and associated alterations. Low-field MRI, benefiting from reduced susceptibility effects, is a promising alternative for lung imaging to limit radiations absorbed by patients during CT examinations, which also have limited capability to assess functional alterations. The aim of this investigative study was to explore the functional abnormalities that free-breathing 0.55 T MRI in combination with the phase-resolved functional lung (PREFUL) analysis could identify in patients with persistent symptoms after COVID-19 infection. MATERIALS AND METHODS: Seventy-four COVID-19 patients and 8 healthy volunteers were prospectively scanned in free-breathing with a balanced steady-state free-precession sequence optimized at 0.55 T, 5 months postinfection on average. Normalized perfusion (Q), fractional ventilation (FV), and flow-volume loop correlation (FVLc) maps were extracted with the PREFUL technique. Q, FV, and FVLc defects as well as defect overlaps between these metrics were quantified. Morphological turbo-spin-echo images were also acquired, and the extent of abnormalities was scored by a board-certified radiologist. To investigate the functional correlates of persistent symptoms, a recursive feature elimination algorithm was applied to find the most informative variables to detect the presence of persistent symptoms with a logistic regression model and a cross-validation strategy. All MRI metrics, sex, age, body mass index, and the presence of preexisting lung conditions were included. RESULTS: The most informative variables to detect persistent symptoms were the percentage of concurrent Q and FVLc defects and of areas free of those defects. A detection accuracy of 71.4% was obtained with these 2 variables when fitting the model on the entire dataset. Although none of the single variables differed between patients with and without persistent symptoms ( P > 0.05), the combined score of these 2 variables did ( P < 0.02). This score also showed a consistent increase from healthy volunteers (7.7) to patients without persistent symptoms (8.2) and with persistent symptoms (8.6). The morphological abnormality score showed poor correlation with the functional parameters. CONCLUSIONS: Functional pulmonary examinations using free-breathing 0.55 T MRI with PREFUL analysis revealed potential quantitative markers of impaired lung function in patients with persistent symptoms after COVID-19 infection, potentially complementing morphologic imaging. Future work is needed to explore the translational relevance and clinical implication of these findings.

Detection of SARS-CoV-2-independent immunoregulatory activity of COVID-19 convalescent plasma.

BACKGROUND: Convalescent plasma has emerged as a potential specific treatment for coronavirus disease 2019 (COVID-19), since it contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. Several studies are currently investigating the efficacy of convalescent plasma for treatment of COVID-19, with a focus on neutralizing antibodies. However, there is little information on whether convalescent plasma may contain additional immunoregulatory constituents produced by the blood donor during convalescence. Therefore, using a standardized whole blood assay employing synthetic toll-like receptor (TLR) ligands, we have investigated the immunoregulatory capacity of convalescent plasma in direct comparison to ABO-matched allogeneic control plasma. STUDY DESIGN AND METHODS: Whole blood samples from healthy blood donors were collected, and autologous plasma was replaced by convalescent plasma or ABO-matched control plasma. Standardized innate immune triggering and monitoring was performed by adding different TLR ligands (Pam3CsK4 [TLR1/2], HKLM [TLR2], LPS [TLR4], flagellin [TLR5], ssRNA40 [TLR8], imiquimod [TLR7], and FSL-1 [TLR2/6]) and subsequent quantitative analysis of pro- and anti-inflammatory cytokines (IP-10, IL-1ß, TNF-α, MCP-1, IL-6, IL-10, and IFN-γ) by cytometric bead array. Negative controls included unstimulated samples as well as samples spiked with autologous plasma. RESULTS: COVID-19 convalescent plasma (CCP) significantly decreased pro-inflammatory cytokines production triggered by different TLR ligands in healthy donors as compared with healthy control plasma. IL-6, MCP-1, and IFN-γ represented the cytokines that are most frequently downregulated by convalescent plasma. CONCLUSION: Our experiments reveal a potential novel, SARS-CoV-2-independent immunomodulatory activity of CCP, which may be beneficial for COVID-19 patients.

Successful treatment of COVID-19 infection with convalescent plasma in B-cell-depleted patients may promote cellular immunity.

Treatment with convalescent plasma has been shown to be safe in coronavirus disease in 2019 (COVID-19) infection, although efficacy reported in immunocompetent patients varies. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody-producing B cells, such as those treated with rituximab (anti-CD20) for hematological malignancies, lack a fundamental part of their adaptive immunity. Treatment with convalescent plasma appears to be of general benefit in this particularly vulnerable cohort. We analyzed clinical course and inflammation markers of three B-cell-depleted patients suffering from COVID-19 who were treated with convalescent plasma. In addition, we measured serum antibody levels as well as peripheral blood CD38/HLA-DR-positive T-cells ex vivo and CD137-positive T-cells after in vitro stimulation with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides in these patients. We observed that therapy with convalescent plasma was effective in all three patients and analysis of CD137-positive T-cells after stimulation with SARS-CoV-2 peptides showed an increase in peptide-specific T-cells after application of convalescent plasma. In conclusion, we here demonstrate efficacy of convalescent plasma therapy in three B-cell-depleted patients and present data that suggest that while application of convalescent plasma elevates systemic antibody levels only transiently, it may also boost specific T-cell responses.

Diagnostic performance of four SARS-CoV-2 antibody assays in patients with COVID-19 or with bacterial and non-SARS-CoV-2 viral respiratory infections.

SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly vulnerable patients. So far, data on cross-reactivity of SARS-CoV-2 antibody assays is limited. Here, we compared four enzyme-linked immunosorbent assays (ELISAs; Vircell SARS-CoV-2 IgM/IgA and IgG, Euroimmun SARS-CoV-2 IgA and IgG) for detection of anti-SARS-CoV-2 antibodies in 207 patients with COVID-19, 178 patients with serological evidence of different bacterial infections, 107 patients with confirmed viral respiratory disease, and 80 controls from the pre-COVID-19 era. In COVID-19 patients, the assays showed highest sensitivity in week 3 (Vircell-IgM/A and Euroimmun-IgA: 78.9% each) and after week 7 (Vircell-IgG: 97.9%; Euroimmun-IgG: 92.1%). The antibody indices were higher in patients with fatal disease. In general, IgM/IgA assays had only limited or no benefit over IgG assays. In patients with non-SARS-CoV-2 respiratory infections, IgG assays were more specific than IgM/IgA assays, and bacterial infections were associated with more false-positive results than viral infections. The specificities in bacterial and viral infections were 68.0 and 81.3% (Vircell-IgM/IgA), 84.8 and 96.3% (Euroimmun-IgA), 97.8 and 86.0% (Vircell-IgG), and 97.8 and 99.1% (Euroimmun-IgG), respectively. Sera from patients positive for antibodies against Mycoplasma pneumoniae, Chlamydia psittaci, and Legionella pneumophila yielded particularly high rates of unspecific false-positive results in the IgM/IgA assays, which was revealed by applying a highly specific flow-cytometric assay using HEK 293 T cells expressing the SARS-CoV-2 spike protein. Positive results obtained with anti-SARS-CoV-2 IgM/IgA ELISAs require careful interpretation, especially if there is evidence for prior bacterial respiratory infections.

IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to an adaptive immune response in the host and the formation of anti-SARS-CoV-2 specific antibodies. While IgG responses against SARS-CoV-2 have been characterized quite well, less is known about IgA. IgA2 activates immune cells and induces inflammation and neutrophil extracellular trap (NET) formation which may contribute to organ injury and fatal outcome in SARS-CoV-2-infected patients. SARS-CoV-2 spike protein specific antibody levels were measured in plasma samples of 15 noninfected controls and 82 SARS-CoV-2-infected patients with no or mild symptoms, moderate symptoms (hospitalization) or severe disease (intensive care unit, ICU). Antibody levels were compared to levels of C-reactive protein (CRP) and circulating extracellular DNA (ecDNA) as markers for general inflammation and NET formation, respectively. While levels of SARS-CoV-2-specific IgG were similar in all patient groups, IgA2 antibodies were restricted to severe disease and showed the strongest discrimination between nonfatal and fatal outcome in patients with severe SARS-CoV-2 infection. While anti-SARS-CoV-2 IgG and IgA2 levels correlated with CRP levels in severely diseased patients, only anti-SARS-CoV-2 IgA2 correlated with ecDNA. These data suggest that the formation of anti-SARS-CoV-2 IgA2 during SARS-CoV-2 infection is a marker for more severe disease related to NET formation and poor outcome.

Validation of a SARS-CoV-2 RNA RT-PCR assay for high-throughput testing in blood of COVID-19 convalescent plasma donors and patients.

BACKGROUND: The frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia in blood donors is uncertain. Thus, assays for SARS-CoV-2 RNA detection in blood, validated on commercially available polymerase chain reaction (PCR) systems, are required to allow a good comparability of data. STUDY DESIGN AND METHODS: The cobas SARS-CoV-2 dual-target reverse transcriptase PCR (RT-PCR) assay, licensed for respiratory swab SARS-CoV-2 RNA testing, was validated for detection of viral RNA in blood. For the validation panel, SARS-CoV-2-positive plasma samples were prepared by spiking SARS-CoV-2-positive respiratory specimens in negative human plasma. The 95% limit of detection (LOD95) was determined by probit analysis. For clinical validation, coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donors and patients with COVID-19 with a severe disease course treated in an intensive care unit (ICU) were included. RESULTS: The validation of the SARS-CoV-2 RT-PCR assay for blood demonstrated high sensitivity and specificity and intra- and inter-assay precision and efficiency. The LOD95 for SARS-CoV-2 RNA was 5.0 genome copies/mL (95% confidence interval [CI], 3.3-12 copies/mL) for target 1 and 4.3 genome copies/mL (95% CI, 2.9-10 copies/mL) for target 2. In a cohort of 39 CCP donors with 66 CCP donations no SARS-CoV-2 RNA in plasma was detected. Screening of 25 blood samples of 19 ICU patients with COVID-19 showed six positive results for SARS-CoV-2 RNA in at least one target of the assay. CONCLUSION: The SARS-CoV-2 RNA assay, only licensed for respiratory swabs, performed on a PCR system for high-throughput testing, showed a good assay performance for blood testing.