Usefulness of real-time RT-PCR to understand the kinetics of SARS-CoV-2 in blood: a prospective study.

BackgroundSARS-CoV-2 viral load and kinetics assessed in serial blood samples from hospitalised COVID-19 patients by RT-PCR are poorly understood. MethodsWe conducted an observational, prospective case series study in hospitalised COVID-19 patients. Clinical outcome data (Intensive Care Unit admission and mortality) were collected from all patients until discharge. Viremia was determined longitudinally during hospitalisation, in plasma and serum samples using two commercial and standardised RT-PCR techniques approved for use in diagnosis of SARS-CoV-2. Viral load (copies/mL and log10) was determined with quantitative TaqPathCOVID-19 test. ResultsSARS-CoV-2 viremia was studied in 57 hospitalised COVID-19 patients. Persistent viremia (PV) was defined as two or more quantifiable viral loads detected in blood samples (plasma/serum) during hospitalisation. PV was detected in 16 (28%) patients. All of them, except for one who rapidly progressed to death, cleared viremia during hospitalisation. Poor clinical outcome occurred in 62.5% of patients with PV, while none of the negative patients or those with sporadic viremia presented this outcome (p<0.0001). Viral load was significantly higher in patients with PV than in those with Sporadic Viremia (p<0.05). Patients presented PV for a short period of time: median time from admission was 5 days (Range=2-12) and 4.5 days (Range=2-8) for plasma and serum samples, respectively. Similar results were obtained with all RT-PCR assays for both types of samples. ConclusionsDetection of persistent SARS-CoV-2 viremia, by real time RT-PCR, expressed as viral load over time, could allow identifying hospitalised COVID-19 patients at risk of poor clinical outcome. HighlightsO_LICommercial RT-PCR techniques could be used to monitor SARS-CoV-2 viremia kinetics. C_LIO_LISARS-CoV-2 persistent viremia is related with poor outcome in COVID-19 patient. C_LIO_LISARS-Cov-2 viremia kinetics could be used as a biomarker of poor prognosis. C_LIO_LIPlasma samples are the best choice for analysis of SARS-CoV-2 viremia kinetics. C_LI

SARS-CoV-2 viremia precedes an IL6 response in severe COVID-19 patients: results of a longitudinal prospective cohort

BackgroundInterleukin 6 (IL6) levels and SARS-CoV-2 viremia have been correlated with COVID-19 severity. The association over time between them has not been assessed in a prospective cohort. Our aim was to evaluate the relationship between SARS-CoV-2 viremia and time evolution of IL6 levels in a COVID-19 prospective cohort. MethodsSecondary analysis from a prospective cohort including COVID-19 hospitalized patients from Hospital Universitario La Princesa between November 2020 and January 2021. Serial plasma samples were collected from admission until discharge. Viral load was quantified by Real-Time Polymerase Chain Reaction and IL6 levels with an enzyme immunoassay. To represent the evolution over time of both variables we used the graphic command twoway of Stata. ResultsA total of 57 patients were recruited, with median age of 63 years (IQR [53-81]), 61.4% male and 68.4% caucasian. The peak of viremia appeared shortly after symptom onset in patients with persistent viremia (more than 1 sample with >1.3 log10 copies/ml) and also in those with at least one IL6>30 pg/ml, followed by a progressive increase in IL6 around 10 days later. Persistent viremia in the first week of hospitalization was associated with higher levels of IL6. Both IL6 and SARS-CoV-2 viral load were higher in males, with a quicker increase with age. ConclusionsIn those patients with worse outcomes, an early peak of SARS-CoV-2 viral load precedes an increase in IL6 levels. Monitoring SARS-CoV-2 viral load during the first week after symptom onset may be helpful to predict disease severity in COVID-19 patients.

Detection of SARS-Cov-2 RNA in serum is associated with increased mortality risk in hospitalized COVID-19 patients.

BackgroundCOVID-19 has overloaded national health services worldwide. Thus, early identification of patients at risk of poor outcomes is critical. Our objective was to analyse SARS-CoV-2 RNA detection in serum as a severity biomarker in COVID-19. Methods and FindingsRetrospective observational study including 193 patients admitted for COVID-19. Detection of SARS-CoV-2 RNA in serum (CoVemia) was performed with samples collected at 48-72 hours of admission by two techniques from Roche and Thermo Fischer Scientific (TFS). Main outcome variables were mortality and need for ICU admission during hospitalization for COVID-19. CoVemia was detected in 50-60% of patients depending on technique. The correlation of Ct in serum between both techniques was good (intraclass correlation coefficient: 0.612; p < 0.001). Patients with CoVemia were older (p = 0.006), had poorer baseline oxygenation (PaO2/FiO2; p < 0.001), more severe lymphopenia (p < 0.001) and higher LDH (p < 0.001), IL-6 (p = 0.021), C-reactive protein (CRP; p = 0.022) and procalcitonin (p = 0.002) serum levels. We defined "relevant CoVemia" when detection Ct was < 34 with Roche and < 31 for TFS. These thresholds had 95% sensitivity and 35 % specificity. Relevant CoVemia predicted death during hospitalization (OR 9.2 [3.8 - 22.6] for Roche, OR 10.3 [3.6 - 29.3] for TFS; p < 0.001). Cox regression models, adjusted by age, sex and Charlson index, identified increased LDH serum levels and relevant CoVemia (HR = 9.87 [4.13-23.57] for TFS viremia and HR = 7.09 [3.3-14.82] for Roche viremia) as the best markers to predict mortality. ConclusionsCoVemia assessment at admission is the most useful biomarker for predicting mortality in COVID-19 patients. CoVemia is highly reproducible with two different techniques (TFS and Roche), has a good consistency with other severity biomarkers for COVID-19 and better predictive accuracy. AUTHOR SUMMARYCOVID-19 shows a very heterogeneous clinical picture. In addition, it has overloaded national health services worldwide. Therefore, early identification of patients with poor prognosis is critical to improve the use of limited health resources. In this work, we evaluated whether baseline SARS-CoV2 RNA detection in blood (CoVemia) is associated with worse outcomes. We studied almost 200 patients admitted to our hospital and about 50-60% of them showed positive CoVemia. Patients with positive CoVemia were older and had more severe disease; CoVemia was also more frequent in patients requiring admission to the ICU. Moreover, we defined "relevant CoVemia", as the amount of viral load that better predicted mortality obtaining 95% sensitivity and 35% specificity. In addition, relevant CoVemia was a better predictor than other biomarkers such as LDH, lymphocyte count, interleukin-6, and indexes used in ICU such as qSOFA and CURB65. In summary, detection of CoVemia is the best biomarker to predict death in COVID-19 patients. Furthermore, it is easy to be implemented and is reproducible with two techniques (Roche and Thermo Fisher Scientific) that are currently used for diagnosis in nasopharyngeal swabs samples.

Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis

Presence of SARS-CoV-2 RNA in serum (viraemia) in COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate the ability of two commercial reverse real-time-PCR (rRT-PCR) kits, cobas(R) SARS-CoV-2 (Cobas(R) test) and TaqPath COVID-19 CE-IVD RT-PCR Kit (Taqpath test), to detect viraemia in COVID-19 patients and their implementation as routine diagnosis in microbiology laboratory. This retrospective cohort study was conducted with 203 adult patients admitted to Hospital Universitario de La Princesa, (89 Intensive Care Unit and 114 ward) with at least one serum sample collected in the first 48 hours from admission. A total 265 serum samples were included for study. Evaluation of both rRT-PCR techniques was performed comparing with the gold standard, a Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit; considering at least one target as a positive result. Comparison of Cobas(R) test and Taqpath test with the gold standard method, showed high values of specificity (93.75 and 92.19 respectively) and Positive Predictive Value (92.92 and 99.88 respectively). Nevertheless, sensitivity (53.72 and 73.63 respectively) and Negative Predictive Value (32.53 and 42.99 respectively) were lower; Kappa values were 0.35 for cobas(R) test and 0.56 for Taqpath test. For both techniques, differences of viraemia detection between the ICU and non-ICU patients were significant (p[≤]0.001). Consequently, SARS-CoV-2 viraemia positive results obtained by both rRT-PCR should be considered good tools and may help in handling COVID-19 patients. Moreover, these methods could be easily integrated in the routine laboratory COVID-19 diagnosis and may open new strategies based on an early COVID-19 treatment.

SARS-Cov-2 cysteine-like protease (Mpro) is immunogenic and can be detected in serum and saliva of COVID-19-seropositive individuals

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2 specific antibodies are based on serum detection of antibodies to either the viral spike glycoprotein (the major target for neutralising antibodies) or the viral nucleocapsid protein that are known to be highly immunogenic in other coronaviruses. Conceivably, exposure of antigens released from infected cells could stimulate antibody responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other non-structural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titre IgG, IgM and IgA antibody responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher antibody titres in these assays associated with more severe disease and no cross-reactive antibodies against prior betacoronavirus were found. Remarkably, IgG antibodies specific for Mpro and other SARS-CoV-2 antigens can also be detected in saliva. In conclusion, Mpro is a potent antigen in infected patients that can be used in serological tests and its detection in saliva could be the basis for a rapid, non-invasive test for COVID-19 seropositivity.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19

BackgroundSARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the pathogenesis and outcome of COVID-19. We aimed to investigate which specific responses from either cellular or humoral immunity associate to severity and progression of COVID-19. MethodsA cohort of 276 patients classified in mild, moderate and severe, was studied. Peripheral blood lymphocyte subpopulations were quantified by flow cytometry, and immunoglobulins and complement proteins by nephelometry. ResultsAt admission, dramatic lymphopenia of T, B and NK cells associated to severity. However, only the proportion of B cells increased, while T and NK cells appeared unaffected. Accordingly, the number of plasma cells and circulating follicular helper T cells (cTfh) increased, but levels of IgM, IgA and IgG were unaffected. When degrees of severity were considered, IgG was lower in severe patients, suggesting an IgG consumption by complement activation or antibody-dependent cellular cytotoxicity (ADCC). Activated CD56-CD16+ NK-cells, which mediate ADCC, were increased. Regarding complement, C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, compared to healthy donors. Moreover, IgG and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. ConclusionOur study provides important clues to understand the immune response observed in COVID-19 patients, which is probably related to viral clearance, but also underlies its pathogenesis and severity. This study associates for the first time COVID-19 severity with an imbalanced humoral immune response characterized by excessive consumption of IgG and C4, identifying new targets for therapeutic intervention.

Differential Redistribution of Activated Monocyte and Dendritic Cell Subsets to the Lung Associates with Severity of COVID-19

The SARS-CoV-2 is responsible for the pandemic COVID-19 in infected individuals, who can either exhibit mild symptoms or progress towards a life-threatening acute respiratory distress syndrome (ARDS). It is known that exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. With the aim to improve the knowledge in this area, we developed a cross-sectional study, in which we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood of COVID-19 patients with different clinical severity in comparison with healthy control individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Collectively, our results suggest that inflammatory transitional and non-classical monocytes preferentially migrate from blood to lungs in patients with severe COVID-19. CD1c+ conventional dendritic cells also followed this pattern, whereas CD141+ conventional and CD123hi plasmacytoid dendritic cells were depleted from blood but were absent in the lungs. Thus, this study increases the knowledge on the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies to fight SARS-CoV-2 infection. Single-sentence summaryDepletion from the blood and differential activation patterns of inflammatory monocytes and CD1c+ conventional dendritic cells associate with development of ARDS in COVID-19 patients.