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J Infect Dev Ctries ; 15(5): 653-656, 2021 05 31.
Article in English | MEDLINE | ID: covidwho-1262632


Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford-AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

COVID-19 Vaccines/immunology , COVID-19/immunology , Immunity, Humoral/drug effects , Antibodies, Neutralizing/immunology , COVID-19/prevention & control , Case-Control Studies , Female , Humans , Male , SARS-CoV-2
Eur J Clin Invest ; 51(6): e13501, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1054522


BACKGROUND: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease). METHODS: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science. RESULTS: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively). CONCLUSION: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.

COVID-19/blood , RNA, Viral/blood , Real-Time Polymerase Chain Reaction/methods , Aged , Ambulatory Care , Female , Humans , Intensive Care Units , Male , Middle Aged , Patients' Rooms , Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Severity of Illness Index
Crit Care ; 24(1): 691, 2020 12 14.
Article in English | MEDLINE | ID: covidwho-977684


BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

COVID-19/complications , RNA, Viral/analysis , Viral Load/immunology , Adult , Aged , Biomarkers/analysis , Biomarkers/blood , COVID-19/blood , Chi-Square Distribution , Critical Illness , Female , Humans , Male , Middle Aged , Multivariate Analysis , Polymerase Chain Reaction/methods , RNA, Viral/blood , Statistics, Nonparametric