Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies

ObjectivesDetection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection.MethodsA cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61).ResultsSpecificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79–95% agreement with the presence of neutralizing antibodies.ConclusionsThe BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.     

新型冠状病毒眼部传播可能性及门诊应对措施探讨

新型冠状病毒肺炎（coronavirus disease 2019, COVID-19）自2019年12月在我国湖北省爆发以来,感染人数迅速增长,并构成全球大流行。COVID-19的病原体是SARS-CoV-2（severe acute respiratory syndrome coronavirus 2）,为冠状病毒β属的单股正链RNA病毒,与SARS-CoV具有79%的基因组同源性。已有学者发现确诊COVID-19的患者泪液及结膜分泌物病毒核酸检测阳性,且有其以结膜炎为首发症状的报道,病毒通过眼部入侵可能与鼻泪管的解剖结构、 SAα2,3 Gal（the sialic acid α2,3-galactose sialyloligosaccharides）受体及血管紧张素转换酶2（angiotensin converting enzyme 2, ACE2）受体的分布有关。眼科门诊存在中老年人比重较大、诊疗中医生与患者距离较近、无保护的眼部暴露风险较高等危险因素,采取必要的防护措施对患者及眼科医生的自我保护以及疫情的控制均有重要意义。     

Room-temperature-storable PCR mixes for SARS-CoV-2 detection

ObjectivesA novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks.MethodsHere, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration.ResultsWe found that the freeze-dried PCR mixes with ~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18 ~ 25 °C) for 28 days, and for 14 and 10 days when stored at 37 °C and 56 °C, respectively.ConclusionThe uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.     

SARS-CoV-2 and cancer: the intriguing and informative cross-talk

The COVID-19 pandemic caused by the SARS-CoV-2 virus has significantly disrupted and burdened the diagnostic workup and delivery of care, including transfusion, to cancer patients across the globe. Furthermore, cancer patients suffering from solid tumors or hematologic malignancies were more prone to the infection and had higher morbidity and mortality than the rest of the population. Major signaling pathways have been identified at the intersection of SARS-CoV-2 and cancer cells, often leading to tumor progression or alteration of the tumor response to therapy. The reactivation of oncogenic viruses has also been alluded to in the context and following COVID-19. Paradoxically, certain tumors responded better following the profound infection-induced immune modulation. Unveiling the mechanisms of the virus-tumor cell interactions will lead to a better understanding of the pathophysiology of both cancer progression and virus propagation. It would be challenging to monitor, through the different cancer registries, retrospectively, the response of patients who have been previously exposed to the virus in contrast to those who have not contracted the infection.     

A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity

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Clinical application of SARS-CoV-2 antibody detection and monoclonal antibody therapies against COVID-19

The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2(SARS-CoV-2) specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies(m Abs) in the treatment of coronavirus infectious disease 2019(COVID-19). The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied. Immunoglobulin M(Ig M) appeared earlier and lasted for a short time, while immunoglobulin G(Ig G) appeared later and last...     

Predictors associated with clinical improvement of SARS-CoV-2 pneumonia

BackgroundThere are few agents that have been proven effective for COVID-19. Predicting clinical improvement as well as mortality or severity is very important.ObjectivesThis study aimed to investigate the factors associated with the clinical improvement of COVID-19.MethodsOverall, 74 patients receiving treatment for COVID-19 at Tokyo Medical and Dental University Hospital from April 6th to May 15th, 2020 were included in this study. Clinical improvement was evaluated, which defined as the decline of two levels on a six-point ordinal scale of clinical status or discharge alive from the hospital within 28 days after admission. The clinical courses were particularly investigated and the factors related to time to clinical improvement were analyzed with the log-rank test and the Cox proportional hazard model.ResultsForty-nine patients required oxygen support during hospitalization, 22 patients required invasive mechanical ventilation, and 5 patients required extracorporeal membrane oxygenation. A total of 83% of cases reached clinical improvement. Longer period of time from onset to admission (≥10 days) (HR, 1.057; 95% CI, 1.002–1.114), no hypertension (HR, 2.077; 95% CI, 1.006–4.287), and low D-dimer levels (<1 μg/ml) (HR, 2.372; 95% CI, 1.229–4.576) were confirmed to be significant predictive factors for time to clinical improvement. Furthermore, a lower SARS-CoV-2 RNA copy number was also a predictive factor for clinical improvement.ConclusionsSeveral predictors for the clinical improvement of COVID-19 pneumonia were identified. These results may be important for the management of COVID-19 pneumonia.     

A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity

Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 μg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants.     

A comparison of SARS-CoV-2 nucleocapsid and spike antibody detection using three commercially available automated immunoassays

IntroductionCommercially available serological assays for SARS-CoV-2 detect antibodies to either the nucleocapsid or spike protein. Here we compare the performance of the Beckman-Coulter SARS-CoV-2 spike IgG assay to that of the Abbott SARS-CoV-2 nucleocapsid IgG and Roche Anti-SARS-CoV-2 nucleocapsid total antibody assays. In addition, we document the trend in nucleocapsid and spike antibodies in sequential samples collected from convalescent plasma donors.MethodsPlasma or serum samples from 20 individual SARS-CoV-2 RT-PCR-positive inpatients (n = 172), 20 individual convalescent donors with a previous RT-PCR-confirmed SARS-CoV-2 infection (n = 20), were deemed positive SARS-CoV-2 samples. RT-PCR-negative inpatients (n = 24), and 109 pre-SARS-CoV-2 samples were determined to be SARS-CoV-2 negative. Samples were assayed by the Abbott, Roche, and Beckman assays.ResultsAll three assays demonstrated 100% specificity. Abbott, Beckman, and Roche platforms had sensitivities of 98%, 93%, and 90% respectively, with the difference in sensitivity attributed primarily to samples from immunocompromised patients. After the exclusion of samples immunocompromised patients, all assays exhibited ≥ 95% sensitivity. In sequential samples collected from the same individuals, the Roche nucleocapsid antibody assay demonstrated continually increasing signal intensity, with maximal values observed at the last time point examined. In contrast, the Beckman spike IgG antibody signal peaked between 14 and 28 days post positive SARS-CoV-2 PCR and steadily declined in subsequent samples. Subsequent collections 51–200 days (median of 139 days) post positive SARS-CoV-2 RT-PCR from five inpatients and five convalescent donors revealed that spike and nucleocapsid antibodies remained detectable for several months after confirmed infection.ConclusionsThe three assays are sensitive and specific for SARS-CoV-2 antibodies. Nucleocapsid and spike antibodies were detectable for up to 200 days post-positive SARS-CoV-2 PCR but demonstrated markedly different trends in signal intensity.     

SARS-CoV-2 antibody changes in patients receiving COVID-19 convalescent plasma from normal and vaccinated donors

Vaccination has been shown to stimulate remarkably high antibody levels in donors who have recovered from COVID-19. Our objective was to measure patient antibody levels before and after transfusion with COVID-19 Convalescent Plasma (CCP) and compare the antibody levels following transfusion of CCP from vaccinated and nonvaccinated donors. Plasma samples before and after transfusion were obtained from 25 recipients of CCP and COVID-19 antibody levels measured. Factors that effect changes in antibody levels were examined. In the 21 patients who received CCP from nonvaccinated donors, modest increases in antibody levels were observed. Patients who received two units were more likely to seroconvert than those receiving just one unit. The strongest predictor of changes in patient antibody level was the CCP dose, calculated by the unit volume multiplied by the donor antibody level. Using patient plasma volume and donor antibody level, the post-transfusion antibody level could be predicted with reasonable accuracy(R²> 0.90). In contrast, the 4 patients who received CCP from vaccinated donors all had dramatic increases in antibody levels following transfusion of a single unit. In this subset of recipients, antibody levels observed after transfusion of CCP were comparable to those seen in donors who had fully recovered from COVID-19. If available, CCP from vaccinated donors with very high antibody levels should be used. One unit of CCP from vaccinated donors increases patient antibody levels much more than 1 or 2 units of CCP from unvaccinated donors.     

Evaluating red blood cell distribution width from community blood tests as a predictor of hospitalization and mortality in adults with SARS-CoV-2: a cohort study

BackgroundRed blood cell distribution width (RDW) has been assessed during COVID-19 patient hospitalization, however, further research should be done to evaluate RDW from routine community blood tests, before infection, as a risk factor for COVID-19 related hospitalization and mortality.Patients and methodsRDW was measured as a predictor along with age, sex, chronic illnesses, and BMI in logistic regressions to predict hospitalization and mortality. Hospitalization and mortality odds ratios (ORs) were estimated with 95% confidence intervals (CI). RDW was evaluated separately as continuous and discrete (High RDW ≥ 14.5) variables.ResultsFour thousand one hundred and sixty-eight patients were included in this study, where 824 patients (19.8%) had a high RDW value ≥14.5% (High RDW: 64.7% were female, mean age 58 years [±22] vs. Normal RDW: 60.2% female, mean age 46 years [±19]). Eight hundred and twenty-nine patients had a hospitalization, where the median time between positive PCR and hospital entry was 5 [IQR 1–18] days. Models were analyzed with RDW (continuous) and adjusted for age, sex, comorbidities, and BMI suggested an OR of 1.242 [95% CI = 1.187–2.688] for hospitalization and an OR of 2.911 [95% CI = 1.928–4.395] for mortality (p < .001). RDW (discrete) with the same adjustments presented an OR of 2.232 [95% CI = 1.853–1.300] for hospitalization and an OR of 1.263 [95% CI = 1.166–1.368] for mortality (p < .001).ConclusionsHigh RDW values obtained from community blood tests are associated with greater odds of hospitalization and mortality for patients with COVID-19.

KEY MESSAGES

• RDW measures before SARS-CoV-2 infection is a predictive factor for hospitalization and mortality.
• RDW threshold of 14.5% provides high sensitivity and specificity for COVID-19 related mortality, comparatively to other blood tests.
• Patient records should be accessed by clinicians for prior RDW results, if available, followed by further monitoring.

     

Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy

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False positive results in severe acute respiratory coronavirus 2 (SARS-CoV-2) rapid antigen tests for inpatients

Severe acute respiratory syndrome coronavirus 2 rapid antigen detection (RAD) test kits are widely used as primary screening test in Japan because rapid diagnosis of coronavirus disease 2019 (COVID-19) is critical for infection control. We report cases with RAD test false-positive results in a ward for patients with disabilities. RAD tests potentially evoke hospital operational risk. It is desirable that performing PCR test appropriately when patients admitted to a medical treatment ward with COVID-19 symptoms instead of RAD test.     

Immunochromatographic test for the detection of SARS-CoV-2 in saliva

We evaluated the rapid immunochromatographic test for severe acute respiratory coronavirus 2 (SARS-CoV-2) antigen detection using 16 saliva specimens collected from 6 COVID-19 hospitalized patients, and detected N-antigen in 4 of 7 RT-PCR positive specimens. This POCT detected SARS-CoV-2 antigen in saliva and would be useful for COVID-19 diagnosis.