Seasonal Increase in Syphilis Positive Testing Rate in Whole Blood Donors 2018-2021

Background/Case Studies: On December 17th 2021 the U.S Food and Drug Administration published a letter to clinical laboratory staff and health care providers detailing a risk of false Rapid Plasma Reagin (RPR) when using the Bio-Rad Laboratories BioPlex 2200 Syphilis Total & RPR kit in people who had received a COVID-19 vaccination. Specifically, this notice stated that Treponema pallidum particle agglutination (TP-PA) assays did not appear to be impacted by this issue. At our institution it has been observed that since 2018, the positivity rate of syphilis screening with negative confirmatory testing has been dramatically increased from previous years. Curiously a striking number of these positives occurred at the end of the year, mainly October through December. Study Design/Methods: All whole blood (WB) donations from 2011-2021 which demonstrated positive syphilis screening with negative confirmatory testing were evaluated. Screening for syphilis was performed using the Beckman Coulter PK TP Microhemagglutination assay with confirmatory testing using CAPTIA Syphilis (T. pallidum)-G. Results/Findings: There were 77 whole blood donations from 59 unique donors screened positive for syphilis with negative confirmatory testing from 2011-2021 (summarized in table 1). A dramatic increase in the unconfirmed syphilis positivity rate was observed in 2018-2021 (mean: 0.439%) compared to 2011-2017 (mean: 0.024%, unpaired t-test p-value: 0.0010), representing an 18-fold increase in positive screens. Of these 77 donations, 8 donors contributed 26 units (median: 3, range: 2-5) between 2018-2021. Three of these 8 donors made several (6, 31, and 85) WB donations with negative syphilis screening prior to becoming positive. The 5 remaining donors switched back and forth between negative and positive over the course their donation history. Conclusion(s): There has been a statistically significant increase in unconfirmed syphilis positivity rate among whole blood donors at our institute since 2018 when compared to the 7 years prior. Additionally, the positivity rate doubled from 2020 to 2021. No changes were made to the testing assay used during this time period that could explain these results. There appears to be an autumnal peak in unconfirmed positives suggesting a possible environmental trigger such as viral infection or influenza/COVID-19 (for the 2021 increase) vaccination. Further investigation would be needed to confirm such a hypothesis. (Table Presented).

COVID-19 Antibody Detection and Assay Performance Using Red Cell Agglutination

Background. Serologic assays detecting antibodies against the SARS-CoV-2 spike or nucleocapsid proteins have been developed to advance our understanding of the prognosis and clinical course of the COVID-19 disease. We recently developed a red cell agglutination-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. This assay uses peptide fragments of the SARS-CoV-2 spike protein to label red cells (C19-kodecytes). We performed a clinical evaluation of this C19-kodecyte assay in COVID-19 convalescent plasma (CCP) donors previously assessed with 2 commercial immunoassays and a virus neutralizing assay. Methods. Red cells were coated with peptide fragments of the SARS-CoV-2 spike protein. We tested plasma samples from 140 CCP donors. The results were compared with those of a virus neutralizing assay and 2 commercial chemiluminescent antibody tests: anti-SARS-CoV-2 Total (IgG, IgM and IgA) assay and anti-SARS-CoV-2 IgG assay (Ortho). Inter-rater agreement between the different assays was measured using Cohen's kappa. Specificity was tested with 150 plasma samples, collected in 2008, more than a decade before the COVID-19 outbreak and with 125 plasma samples, collected in 2020 from consecutive healthy volunteer donors, who tested negative for the Ortho Total assay. Testing was performed using the column agglutination technique commonly employed for blood typing. Results. The area under the ROC curve (AUC) for the C19-kodecyte assay reached 0.95 (95% CI: 0.93 - 0.97) with sensitivity of 92.8% (95% CI: 86.9% - 96.3%) and specificity of 96.3% (95% CI: 93.2% - 98.1%). In almost all of the 40 CCP donors with longitudinal data, the antibody concentration decreased during the follow-up, which ranged from 7 to 44 weeks. In the 140 CCP donors, we compared the C19-kodecyte score to the antibody concentrations from the 2 FDA authorized assays (Ortho Total and Ortho IgG) and the titer in the neutralizing assay. There was a positive relationship between the results of all 4 assays. The Spearman's correlation of our assay was 0.20 with the neutralizing assay (0.49 with IgG, and 0.41 with Total assays). Conclusions. Sensitivity and specificity of the C19-kodecyte assay were within the minimum performance range required by the FDA for EUA authorization of serology tests. The limited correlation in assay reaction strengths suggested that the assays may be influenced by different antibody specificities. Unlike the other 84 FDA authorized serologic tests for SARS-CoV-2, this C19-kodecyte assay is a simple and rapid test that can be easily established in any blood typing laboratory worldwide using its routine setup for column agglutination or tube technique. The technique could vastly improve assay capacity, particularly in resource limited hospital blood banks. Disclosures: Bovin: Kode Biotech: Current Employment, Current holder of stock options in a privately-held company. Henry: Kode Biotech: Current Employment, Current holder of stock options in a privately-held company.

COVID-19 antibodies in convalescent plasma evaluated by the C19-kodecyte (Anti-SARSCoV- 2 Red Cell) Assay

Background/Case Studies: We had developed a simple and rapid red cell agglutination assay for serologic screening of antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated this new C19-kodecyte assay in a cohort of convalescent patients with PCR-confirmed SARS-CoV-2 infection and compared its analytical performance with three established assays. Study Design/Methods: Red cells modified with peptides from SARS-CoV-2 spike protein using the Kode Technology were prepared.We tested plasma samples from 140 convalescent plasma donors and 125 healthy donors that had tested negative for anti-SARS-CoV-2 Total (Ortho). Plasma samples were stored at -30 °C before testing. Testing was performed using the column agglutination technology commonly employed for blood typing. The results were evaluated and compared with the results of a virus neutralization assay and two commercial chemiluminescent antibody tests: Ortho anti-SARS-CoV-2 Total (IgG, IgM and IgA) assay and Ortho anti-SARS-CoV-2 IgG antibody assay. Results/Findings: The median time interval from the onset of symptoms and plasma donation was 94 days (range: 33 - 331 days). Our simple and rapid C19-kodecyte assay detected SARS-CoV-2 antibodies with a specificity of 95.2% and sensitivity of 92.1%. Correlation analysis of measurement data among the different platforms showed a weak to moderate concordance. The Pearson correlation coefficient ranged between 0.21 and 0.28 for C19-kodecyte versus Ortho anti-SARS-CoV-2 Total and Ortho anti-SARS-CoV-2 IgG assays while it was 0.24 between C19-kodecyte and neutralizing titer. Agreements among C19-kodecyte versus Ortho anti-SARS-CoV-2 Total and Ortho anti-SARS-CoV-2 IgG assays were moderate: 0.41 (0.09-0.73) and 0.41 (0.14-0.68), respectively. Conclusions: Sensitivity and specificity of the C19-kodecyte assay are within the estimated range of FDA issued EUA authorized serology tests (88% to 100% sensitivity and 95% to 100% specificity). The low correlation between C19-kodecyte versus Ortho anti-SARSCoV- 2 results and between C19-kodecyte versus neutralizing titer suggests that COVID-19 patients develop a broad antibody repertoire against multiple SARS-CoV-2 proteins and epitopes and different assays detect diverse antibody specificities. Our easily scalable approach using C19-kodecytes can be operated in blood typing laboratories worldwide using common routine setups. The availability of standard blood group serology techniques for SARS-CoV-2 antibody screening could vastly improve assay capacity. This would be particularly useful in developing countries, where dedicated virology and microbiology may be lacking the necessary turnaround capacity.

Serological assays and neutralizing activity in COVID-19 convalescent plasma donors

Background/Case Studies: In the absence of definitive treatment, plasma from convalescent donors is an investigational therapeutic option for COVID-19, caused by the virus SARS-CoV-2. We investigated total (Immunoglobulins A, M, and G), Immunoglobulin G and in vitro neutralizing anti-SARS-CoV-2 antibody titers in COVID-19 Convalescent Plasma (CCP) Donors. Study Design/Methods: We recruited donors who had laboratory evidence of past COVID-19 infection and recovery from COVID-19 for ≥ 28 days. Female donors were tested for HLA antibodies. All donor samples were tested for Anti-SARS-CoV-2 antibodies using the Ortho- Clinical Diagnostics VITROS Total and IgG COVID-19 Antibody Test, a qualitative (semi-quantitative) chemiluminescent CLIA immunoassay (EUA) targeting SARSCoV- 2 antigen subunit 1 [S1] of the spike protein and an in-house plaque reduction neutralizing test (PRNT). Results/Findings: We tested samples from 117 donors, mean age 46.9 ± 14.3 years, F:M=1.25:1;most (92.3%) had mild symptoms of COVID-19. ABO blood groups were O (34.2%), A (35.9%), B (12.8%), and AB (6.8%);30.7% of females had HLA antibodies. At initial presentation, total Anti-SARS-CoV-2 Ab, IgG Ab alone and presence of neutralizing activity were detected in 98.3%, 90.6%, and 82.9% of donors respectively. Neutralizing antibodies were detected in 84.9% (90/106) of IgG Ab positive donors. IgG Ab levels (S/Co) were positively correlated with Total Ab levels (R2=0.66, p <0.0001). IgG Ab levels showed a high degree of correlation with the neutralizing Ab titers (one-way ANOVA p <0.0001). Twentyeight (23.9%) donors presented for repeat donation, after a median interval of 37 days (range 21-72). On repeat presentation, total Ab level increased in 81.5% and IgG Ab decreased in 74.1% of donors;neutralizing Ab titers decreased in 44.4% and remained unchanged in 33.3% of repeat donors. There was no correlation observed between total Ab levels and number of days postsymptom onset (R2 = 0.09). Total Ab and IgG Ab levels were significantly different based on degree of symptomseverity (p = 0.019 and 0.022, respectively), with statistically significant differences between asymptomatic and severe cases (p = 0.041 and 0.030, respectively). There was no correlation between Total Ab, IgG Ab, or neutralizing Ab titers with age, sex, ABO blood group or HLA antibody status. Conclusions: 98% of CCP donors had detectable anti- SARS-CoV-2 antibody, and 83% had neutralizing antibodies. IgG Ab correlated strongly with neutralizing Ab titers. On repeat presentation, neutralizing Ab titers decreased in 44.4%, and remained unchanged in 33%.