Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology.

BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm. METHODS: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. CONCLUSION: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Cirrhosis-Associated RAS-Inflammation-Coagulation Axis Anomalies: Parallels to Severe COVID-19.

(1) Background: Cirrhotic patients have an increased risk for severe COVID-19. We investigated the renin-angiotensin-aldosterone system (RAS), parameters of endothelial dysfunction, inflammation, and coagulation/fibrinolysis in cirrhotic patients and in COVID-19 patients. (2) Methods: 127 prospectively characterized cirrhotic patients (CIRR), along with nine patients with mild COVID-19 (mild-COVID), 11 patients with COVID-19 acute respiratory distress syndrome (ARDS; ARDS-COVID), and 10 healthy subjects (HS) were included in the study. Portal hypertension (PH) in cirrhotic patients was characterized by hepatic venous pressure gradient (HVPG). (3) Results: With increased liver disease severity (Child-Pugh stage A vs. B vs. C) and compared to HS, CIRR patients exhibited higher RAS activity (angiotensin-converting enzyme (ACE), renin, aldosterone), endothelial dysfunction (von Willebrand-factor (VWF) antigen), inflammation (C-reactive protein (CRP), interleukin-6 (IL-6)), and a disturbed coagulation/fibrinolysis profile (prothrombin fragment F1,2, D-dimer, plasminogen activity, antiplasmin activity). Increased RAS activity (renin), endothelial dysfunction (vWF), coagulation parameters (D-dimer, prothrombin fragment F1,2) and inflammation (CRP, IL-6) were significantly altered in COVID patients and followed similar trends from mild-COVID to ARDS-COVID. In CIRR patients, ACE activity was linked to IL-6 (ρ = 0.26; p = 0.003), independently correlated with VWF antigen (aB: 0.10; p = 0.001), and was inversely associated with prothrombin fragment F1,2 (aB: -0.03; p = 0.023) and antiplasmin activity (aB: -0.58; p = 0.006), after adjusting for liver disease severity. (4) Conclusions: The considerable upregulation of the RAS in Child-Pugh B/C cirrhosis is linked to systemic inflammation, endothelial dysfunction, and abnormal coagulation profile. The cirrhosis-associated abnormalities of ACE, IL-6, VWF antigen, and antiplasmin parallel those observed in severe COVID-19.

Long-term follow-up after successful treatment of vaccine-induced prothrombotic immune thrombocytopenia.

BACKGROUND: Cases of ChAdOx1 nCoV-19 (AstraZeneca) vaccinated patients with thrombocytopenia, elevated D-dimer, and elevated platelet factor 4 (PF4) antibody levels with- and without thrombosis have been reported. No recommendations regarding the duration of anticoagulation have been made, because data on the long-term course beyond the first weeks is lacking. OBJECTIVE: To report on the treatment, medical course, and longitudinal follow-up of laboratory parameters in patients with vaccine-induced prothrombotic immune thrombocytopenia (VIPIT). PATIENTS: We followed VIPIT patients with- (n = 3) and without (n = 3) venous thromboembolism fulfilling the aforementioned laboratory criteria. RESULTS: Elevated D-dimer (median: 35.10 µg/ml, range: 17.80-52.70), thrombocytopenia (42 G/l, 20-101), and strong positivity in the platelet factor 4 (PF4)/heparin-enzyme-immunoassay (2.42 optical density [OD], 2.06-3.13; reference range < 0.50) were present in all patients after vaccination (10 days, 7-17). Routine laboratory parameters rapidly improved upon initiation of treatment (comprising therapeutic non-heparin anticoagulation in all patients and high dose immunoglobulins ± corticosteroids in 5 patients). PF4 antibody levels slowly decreased over several weeks. Patients were discharged in good physical health (8 days, 5-13). VIPIT did not recur during follow-up (12 weeks, 8-17). Five of 6 patients fully recovered (in 2 patients thrombosis had resolved, in 1 patient exertional dyspnea persisted). CONCLUSIONS: Remissions without sequelae can be achieved upon rapid initiation of treatment in patients with VIPIT. Platelet factor 4 antibody levels slowly decreased over several weeks but VIPIT did not recur in any of our patients. Continuation of anticoagulation in VIPIT patients at least until PF4 antibody negativity is reached seems reasonable.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

Successful treatment of vaccine-induced prothrombotic immune thrombocytopenia (VIPIT).

Cases of unusual thrombosis and thrombocytopenia after administration of the ChAdOx1 nCoV-19 vaccine (AstraZeneca) have been reported. The term vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) was coined to reflect this new phenomenon. In vitro experiments with VIPIT patient sera indicated that high-dose intravenous immunoglobulins (IVIG) competitively inhibit the platelet-activating properties of ChAdOx1 nCoV-19 vaccine induced antibodies. Here, we report a case of a 62-year-old woman who had received this vaccine and developed VIPIT. She visited the emergency ward because of petechiae and hematomas. In the laboratory work-up, thrombocytopenia, low fibrinogen, elevated D-dimer, and positivity in the platelet factor 4/heparin-enzyme-immunoassay were present. Signs and symptoms of thrombosis were absent. Upon immediate therapy with non-heparin anticoagulation, high-dose IVIG, and prednisolone, laboratory parameters steadily improved and the patient was discharged from hospital without thrombotic complications. We conclude that early initiation of VIPIT treatment results in a swift response without thrombotic complications.

Side-by-Side Comparison of Three Fully Automated SARS-CoV-2 Antibody Assays with a Focus on Specificity.

BACKGROUND: In the context of the COVID-19 pandemic, numerous new serological test systems for the detection of anti-SARS-CoV-2 antibodies rapidly have become available. However, the clinical performance of many of these is still insufficiently described. Therefore, we compared 3 commercial CE-marked, SARS-CoV-2 antibody assays side by side. METHODS: We included a total of 1154 specimens from pre-COVID-19 times and 65 samples from COVID-19 patients (≥14 days after symptom onset) to evaluate the test performance of SARS-CoV-2 serological assays by Abbott, Roche, and DiaSorin. RESULTS: All 3 assays presented with high specificities: 99.2% (98.6-99.7) for Abbott, 99.7% (99.2-100.0) for Roche, and 98.3% (97.3-98.9) for DiaSorin. In contrast to the manufacturers' specifications, sensitivities only ranged from 83.1% to 89.2%. Although the 3 methods were in good agreement (Cohen's Kappa 0.71-0.87), McNemar tests revealed significant differences between results obtained from Roche and DiaSorin. However, at low seroprevalences, the minor differences in specificity resulted in profound discrepancies of positive predictive values at 1% seroprevalence: 52.3% (36.2-67.9), 77.6% (52.8-91.5), and 32.6% (23.6-43.1) for Abbott, Roche, and DiaSorin, respectively. CONCLUSION: We found diagnostically relevant differences in specificities for the anti-SARS-CoV-2 antibody assays by Abbott, Roche, and DiaSorin that have a significant impact on the positive predictive values of these tests.