Measurement of SARS-CoV-2 Antibody Titers Improves the Prediction Accuracy of COVID-19 Maximum Severity by Machine Learning in Non-Vaccinated Patients.

Numerous studies have suggested that the titers of antibodies against SARS-CoV-2 are associated with the COVID-19 severity, however, the types of antibodies associated with the disease maximum severity and the timing at which the associations are best observed, especially within one week after symptom onset, remain controversial. We attempted to elucidate the antibody responses against SARS-CoV-2 that are associated with the maximum severity of COVID-19 in the early phase of the disease, and to investigate whether antibody testing might contribute to prediction of the disease maximum severity in COVID-19 patients. We classified the patients into four groups according to the disease maximum severity (severity group 1 (did not require oxygen supplementation), severity group 2a (required oxygen supplementation at low flow rates), severity group 2b (required oxygen supplementation at relatively high flow rates), and severity group 3 (required mechanical ventilatory support)), and serially measured the titers of IgM, IgG, and IgA against the nucleocapsid protein, spike protein, and receptor-binding domain of SARS-CoV-2 until day 12 after symptom onset. The titers of all the measured antibody responses were higher in severity group 2b and 3, especially severity group 2b, as early as at one week after symptom onset. Addition of data obtained from antibody testing improved the ability of analysis models constructed using a machine learning technique to distinguish severity group 2b and 3 from severity group 1 and 2a. These models constructed with non-vaccinated COVID-19 patients could not be applied to the cases of breakthrough infections. These results suggest that antibody testing might help physicians identify non-vaccinated COVID-19 patients who are likely to require admission to an intensive care unit.

Association of the Serum Levels of the Nucleocapsid Antigen of SARS-CoV-2 With the Diagnosis, Disease Severity, and Antibody Titers in Patients With COVID-19: A Retrospective Cross-Sectional Study.

Background: Several types of laboratory tests for COVID-19 have been established to date; however, the clinical significance of the serum SARS-CoV-2 nucleocapsid (N) antigen levels remains to be fully elucidated. In the present study, we attempted to elucidate the usefulness and clinical significance of the serum N antigen levels. Methods: We measured the serum N antigen levels in 391 serum samples collected from symptomatic patients with a confirmed diagnosis of COVID-19 and 96 serum samples collected from patients with non-COVID-19, using a fully automated chemiluminescence immunoassay analyzer. Results: Receiver operating characteristic analysis identified the optimal cutoff value of the serum N antigen level (cutoff index, based on Youden's index) as 0.255, which yielded a sensitivity and specificity for the diagnosis of COVID-19 of 91.0 and 81.3%, respectively. The serum N antigen levels were significantly higher in the patient groups with moderate and severe COVID-19 than with mild disease. Moreover, a significant negative correlation was observed between the serum N antigen levels and the SARS-CoV-2 IgG antibody titers, especially in patients with severe COVID-19. Conclusion: Serum N antigen testing might be useful both for the diagnosis of COVID-19 and for obtaining a better understanding of the clinical features of the disease.

Response kinetics of different classes of antibodies to SARS-CoV2 infection in the Japanese population: The IgA and IgG titers increased earlier than the IgM titers.

To better understand the immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with COVID-19, it is important to investigate the kinetics of the antibody responses and their associations with the clinical course in different populations, since there seem to be considerable differences between Western and Asian populations in the clinical features and spread of COVID-19. In this study, we serially measured the serum titers of IgM, IgG and IgA antibodies generated against the nucleocapsid protein (NCP), S1 subunit of the spike protein (S1), and receptor-binding domain in the S1 subunit (RBD) of SARS-CoV-2 in Japanese individuals with COVID-19. Among the IgM, IgG, and IgA antibodies, IgA antibodies against all of the aforementioned viral proteins were the first to appear after the infection, and IgG and/or IgA seroconversion often preceded IgM seroconversion. In regard to the timeline of the antibody responses to the different viral proteins (NCP, S1 and RBD), IgA against NCP appeared than IgA against S1 or RBD, while IgM and IgG against S1 appeared earlier than IgM/IgG against NCP or RBD. The IgG responses to all three viral proteins and responses of all three antibody classes to S1 and RBD were sustained for longer durations than the IgA/IgM responses to all three viral proteins and responses of all three antibody classes to NCP, respectively. The seroconversion of IgA against NCP occurred later and less frequently in patients with mild COVID-19. These results suggest possible differences in the antibody responses to SARS-CoV-2 antigens between the Japanese and Western populations.

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan.

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.

Time course of the sensitivity and specificity of anti-SARS-CoV-2 IgM and IgG antibodies for symptomatic COVID-19 in Japan.

The accurate and prompt diagnosis of SARS-CoV-2 infection is required for the control and treatment of the coronavirus infection disease 2019 (COVID-19). In this study, we aimed to investigate the time courses of the anti-severe acute corona respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and IgG titers and to evaluate the sensitivity and specificity of such tests according to the specific day after the onset of COVID-19 among a patient population in Japan. We measured the titers of SARS-CoV-2 IgM and IgG in sera from 105 subjects, including 26 symptomatic COVID-19 patients, using chemiluminescent immunoassay (CLIA) methods utilizing magnetic beads coated with SARS-CoV-2 nucleocapsid protein and spike protein. The results of a ROC analysis suggested the possibility that the cutoff values in Japan might be lower than the manufacturer's reported cutoff (10 AU/mL): 1  AU/mL for IgM and 5  AU/mL for IgG. The sensitivity of the test before Day 8 after symptom onset was less than 50%; at Days 9-10, however, we obtained a much higher sensitivity of 81.8% for both IgM and IgG. At 15 days or later after symptom onset, the SARS-CoV-2 IgG test had a sensitivity of 100%. These results suggest that if the number of days since disease onset is taken into consideration, these antibody tests could be very useful for the diagnosis of COVID-19 and similar diseases.

Chloroquine and hydroxychloroquine provoke arrhythmias at concentrations higher than those clinically used to treat COVID-19: A simulation study.

The risk of fatal arrhythmias is the major concern for using chloroquine (CQ) or hydroxychloroquine (HCQ) to treat coronavirus disease 2019 (COVID-19), but the reported number of life-threatening arrhythmic events or deaths is relatively small. The objective of this study was to assess the arrhythmogenic risk of these two drugs using a multiscale heart simulation, which allows testing even at high concentrations, including those that cause fatal arrhythmias. We measured the inhibitory action of CQ, HCQ, and HCQ with 30 µM azithromycin (AZ) on six ion currents (fast [INa] and late [INa,L] components of the sodium current, L-type calcium current [ICa,L], rapid [IKr/hERG], and slow [IKs] components of delayed rectifier potassium, and inward rectifier potassium [IK1]) over a wide range of concentrations using the automated patch-clamp system. Using the concentration-inhibition relationship that was thus obtained, we simulated the drug effects while increasing the concentration until the life-threatening arrhythmia, torsade de pointes (TdP), was observed. The obtained threshold concentrations for TdP were 12.5, 35, and 22.5 µM for CQ, HCQ, and HCQ with AZ, respectively. Adding therapeutic concentrations of mexiletine or verapamil successfully prevented the occurrence of TdP, and verapamil was more effective. CQ, HCQ, and HCQ with AZ thresholds for TdP were larger than both antiviral concentrations that were reported by in vitro experiments and free plasma concentrations that were attained by the clinically used dosage. The current simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Despite the potent in vitro antiviral effect, clinical trials have failed to show the therapeutic effects of chloroquine (CQ) and hydroxychloroquine (HCQ)/azithromycin (AZ) to treat coronavirus disease 2019. Torsadogenic potentials may limit the dosage of these drugs, but the reported incidence of fatal arrhythmias is rare. WHAT QUESTION DID THIS STUDY ADDRESS? Our objective was to assess the arrhythmogenicity of CQ and HCQ/AZ over a wide range of drug concentrations using a multiscale heart simulation. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Our study showed that CQ and HCQ/AZ do not induce fatal arrhythmias even at concentrations much higher than in vitro antiviral half-maximal effective concentration (EC50 ) values at which QT prolongation exceeds 150 ms. We also found that estimated free plasma concentrations of CQ and HCQ/AZ achieved by currently used dosing protocols are lower than the antiviral EC50 for these drugs. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Our simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ.