A rapid, easy, and scalable whole blood monocyte CD169 assay for outpatient screening during SARS-CoV-2 outbreak, and potentially other emerging disease outbreaks.

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients. Methods: Patients (n = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: We observed 98% and 100% sensitivity in early-stage (n = 52) and asymptomatic patients (n = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups. Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during COVID-19 and if monocytes and macrophages could be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Monocytes and monocyte-derived macrophages (MDMs) from COVID-19 patients and controls were infected with SARS-CoV-2 and extensively investigated with immunofluorescence, viral RNA extraction and quantification, and total RNA extraction followed by reverse-transcription quantitative polymerase chain reaction using specific primers, supernatant cytokines (interleukins 6, 10, and 1ß; interferon-ß; transforming growth factor-ß1, and tumor necrosis factor-α), and flow cytometry. The effect of M1- vs M2-type or no polarization prior to infection was assessed. RESULTS: SARS-CoV-2 efficiently infected monocytes and MDMs, but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In COVID-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. CONCLUSIONS: SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of COVID-19 progression.SARS-CoV-2 infection of macrophages induces a specific M2 transcriptional program. In Covid-19 patients, monocyte subsets were decreased associated with up-expression of the immunoregulatory molecule CD163 suggesting that SARS-CoV-2 drives immune system for the benefit of Covid-19 disease progression.

A Granulocytic Signature Identifies COVID-19 and Its Severity.

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.

Evaluating ELISA, Immunofluorescence, and Lateral Flow Assay for SARS-CoV-2 Serologic Assays.

BACKGROUND: The SARS-CoV-2 outbreak has emerged at the end of 2019. Aside from the detection of viral genome with specific RT-PCR, there is a growing need for reliable determination of the serological status. We aimed at evaluating five SARS-CoV-2 serology assays. METHODS: An in-house immunofluorescence assay (IFA), two ELISA kits (EUROIMMUN® ELISA SARS-CoV-2 IgG and NovaLisa® SARS-CoV-2 IgG and IgM) and two lateral flow assays (T-Tek® SARS-CoV-2 IgG/IgM Antibody Test Kit and Sure Bio-tech® SARS-CoV-2 IgM/IgG Antibody Rapid Test) were compared on 40 serums from RT-PCR-confirmed SARS-CoV-2 infected patients and 10 SARS-CoV-2 RT-PCR negative subjects as controls. RESULTS: Control subjects tested negative for SARS-CoV-2 antibodies with all five systems. Estimated sensitivities varied from 35.5 to 71.0% for IgG detection and from 19.4 to 64.5% for IgM detection. For IgG, in-house IFA, EuroImmun, T-Tek and NovaLisa displayed 50-72.5% agreement with other systems except IFA vs EuroImmun and T-Tek vs NovaLisa. Intermethod agreement for IgM determination was between 30 and 72.5%. DISCUSSION: The overall intermethod agreement was moderate. This inconsistency could be explained by the diversity of assay methods, antigens used and immunoglobulin isotype tested. Estimated sensitivities were low, highlighting the limited value of antibody detection in CoVID-19. CONCLUSION: Comparison of five systems for SARS-CoV-2 IgG and IgM antibodies showed limited sensitivity and overall concordance. The place and indications of serological status assessment with currently available tools in the CoVID-19 pandemic need further evaluations.

Immune Modulation as a Therapeutic Option During the SARS-CoV-2 Outbreak: The Case for Antimalarial Aminoquinolines.

The rapid spread, severity, and lack of specific treatment for COVID-19 resulted in hasty drug repurposing. Conceptually, trials of antivirals were well-accepted, but twentieth century antimalarials sparked an impassioned global debate. Notwithstanding, antiviral and immunomodulatory effects of aminoquinolines have been investigated in vitro, in vivo and in clinical trials for more than 30 years. We review the mechanisms of action of (hydroxy)chloroquine on immune cells and networks and discuss promises and pitfalls in the fight against SARS-CoV-2, the agent of the COVID-19 outbreak.