OBJECTIVE:We sought to establish the US Department of Veterans Affairs (VA) Veterans Health Administration (VHA) Sequencing for Research Clinical and Epidemiology (SeqFORCE) multilaboratory consortium for SARS-CoV-2 whole-genome sequencing (WGS). METHODS:Clinical criteria were established for sending patient and employee samples from 145 VHA medical centers to 10 VHA clinical laboratories using 4 different WGS platforms. A linked pipeline among laboratories for SARS-CoV-2 clade and lineage interpretation, result transmission to electronic health records, and data storage was developed. RESULTS:The SeqFORCE program went live on July 1, 2021. As of December 15, 2024, 51 307 samples have been analyzed by WGS for SARS-CoV-2. The median participant age was 60 years, 76.6% were male, and 13.5% were inpatients; 96.5% were Delta, Omicron, and Recombinant sublineages; and 78.5% represented SARS-CoV-2 postvaccination samples among patients and staff. CONCLUSIONS:Establishment of VA SeqFORCE enabled national population analysis for use in epidemiologic response and population health policy as well as expanded SARS-CoV-2 sequencing capacity to meet the demand for clinical and public health sequencing. The program consolidated operations using standardized procedures, test setup, analysis, reporting, and tracking. It also improved oversight and governance of VA contributions to global databases, mitigated system inefficiencies, and prepared VHA for future genomic challenges.

ABSTRACT:Polypeptide blood group antigens, many of which are created by single exofacial amino acid substitutions, have varying immunogenicities. Why some amino acid substitutions are more immunogenic than others is little understood. Using AlphaFold2, an artificial intelligence system that predicts 3-dimensional protein structure, along with multiple other structure analysis programs, we investigated protein structure at sites of amino acid substitutions that create 9 clinically significant blood group antigens. Based on structure predictions, the amino acid substitutions that create the 4 most immunogenic of the 9 antigens (K, Jka, Lua, and E) were typically buried or partially buried in rigid, ordered protein regions, usually helices and β-strands. This was reflected by their lower mean relative solvent accessibility (RSA) than the 5 less immunogenic antigens (c, M, Fya, C, and S; 0.13 vs 0.81; P = .003) and higher mean AlphaFold2 confidence score (92.5 vs 48.3; P = .001; scores <50 predict protein disorder). Substitutions creating the 5 least immunogenic antigens (c, Fya, M, C, and S) were all predicted to be in flexible regions with high accessibility, either in surface-accessible loops (C, c) or disordered coils (Fya, M, and S). Scatter plots revealed a positive linear correlation of immunogenicity with confidence score (R2 = 0.826; P = .0007) and percent helix/β-strand in 15-mers centered around the substitution sites (R2 = 0.763; P = .0021) and a negative linear correlation with RSA (R2 = 0.688; P = .0057). Therefore, based on an informatics analysis, the protein secondary and tertiary structures at amino acid substitution sites that create blood group antigens are significant correlates and potential determinants of immunogenicity.

Experimental evidence suggests that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) remains viable within aerosols with a half-life of approximately 3 hours; however, it remains unclear how long airborne SARS-CoV-2 can transmit infection. Whole genome sequencing during an outbreak suggested inroom transmission of SARS-CoV-2 to two patients admitted nearly 2 and 5 hours, respectively, after discharge of an asymptomatic infected patient. These findings suggest that airborne SARS-CoV-2 may transmit infection for over 4 hours, even in a hospital setting. Published by Elsevier Inc. on behalf of Association for Professionals in Infection Control and Epidemiology, Inc.

Experimental evidence suggests that SARS-CoV-2 remains viable within aerosols with a half-life of approximately 1-3 hours, though changes in aerosol microenvironment may shorten viability to minutes. However, it remains unclear how long airborne SARS-CoV-2 can transmit infection. Whole genome sequencing of nasopharyngeal samples obtained from patients on an outbreak unit suggested in-room transmission of the delta variant, AY3 lineage, of SARS-CoV-2 to two patients admitted 1 hour, 43 minutes and 4 hours, 45 minutes after discharge of an asymptomatic infected patient. These findings suggest that airborne SARS-CoV-2 may transmit infection for nearly 5 hours, even in a hospital setting.

Introduction The objective of this study was to describe the correlation between the commercially available assay for anti-S1/RBD IgG and protective serum neutralizing antibodies (nAb) against SARS-CoV-2 in an adult population after SARS-CoV-2 vaccination, and determine if clinical variables impact this correlation. Methods We measured IgG anti-S1/RBD using the IgG-II CMIA assay and nAb IC 50 values against SARS-CoV-2 WA-1 in sera serially collected post-mRNA vaccination in veterans and healthcare workers of the Veterans Affairs Connecticut Healthcare System (VACHS) between December 2020 and January 2022. The correlation between IgG and IC 50 was measured using Pearson correlation. Clinical variables (age, sex, race, ethnicity, prior COVID infection defined by RT-PCR, history of malignancy, estimated glomerular filtration rate (GFR calculated using CKD-EPI equation) were collected by manual chart review. The impact of these clinical variables on the IgG-nAb correlation was analyzed first with univariable regression. Variables with a significance of p < 0.15 were analyzed with forward stepwise regression analysis. Results From 127 sera samples in 100 unique subjects (age 20–93 years; mean 63.83; SD 15.63; 29% female; 67% White), we found a robust correlation between IgG anti-S1/RBD and nAb IC 50 ( R 2 = 0.83, R 2 adj = 0.70, p < 0.0001). Race, ethnicity, and a history of malignancy were not significant on univariable analysis. GFR (p < 0.05) and prior COVID infection (p < 0.001) had a significant impact on the correlation between IgG anti-S1/RBD and nAb IC 50 . Age (p = 0.06) and sex (p = 0.07) trended towards significance on univariable analysis, but were not significant on multivariable regression. Conclusions There was a strong correlation between IgG anti-S1/RBD and nAb IC 50 after SARS-CoV-2 vaccination. Clinical comorbidities, such as prior COVID infection and renal function, impacted this correlation. These results may assist the prediction of post-vaccination immune protection in clinical settings using cost-effective commercial platforms.

BACKGROUND:Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens.STUDY DESIGN AND METHODS:The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity.RESULTS:Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope.CONCLUSION:Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Abstract Background The US Department of Veterans Affairs (VA) created the Sequencing For Research, Clinical, Epidemiology (SEQFORCE) Program in July 2021 to conduct SARS-CoV-2 Whole Genome Sequencing (WGS). Herein, we describe SARS-CoV-2 variants associated with COVID-19 infection, including after bivalent vaccination. Methods Demographics, COVID-19 vaccinations, hospitalizations, SARS-CoV-2 variants, and Charlson comorbidity index variables were extracted from VA data sources from 7/1/21-4/1/23. Eligible respiratory samples required a positive RT-PCR result from any platform with cycle threshold < 30. WGS was performed using 3 different platforms (Clear Labs, Illumina, ThermoFisher) at the 9 laboratories and one analytic pipeline (PraediGene, Bitscopic) using Pangolin and Nextclade. Post-vaccine COVID-19 infection was defined as > 2 weeks after COVID-19 vaccine receipt. Results Over 41,000 samples from 150 VA clinical sites across all geographical locations in the country have been analyzed by 9 SEQFORCE laboratories since July 1, 2021 (Figure 1), including 28,800 after vaccine breakthrough infection (Table 1). Since October 1, 2022, 3,087 patients had SARS-CoV-2 variants characterized including 986 after bivalent vaccination, 1,191 who were fully vaccinated (including Janssen), but not boosted and didn’t receive a bivalent vaccine dose, and 910 who were never vaccinated (Figure 2) according to VA records. Those who received bivalent vaccine were significantly older, male, and had higher Charlson morbidity scores compared to those not receiving a bivalent vaccine. There was no difference in infection rates based on type of bivalent vaccine received. Bivalent vaccine recipients had significantly more XBB and less BA.5 variants, compared to not receiving a bivalent vaccine (p < 0.0001 for both comparisons). Conclusion VA established a SARS-CoV-2 sequencing consortium to track variants for clinical and epidemiological indications. Sample submission was voluntary and therefore may have limited geographic, temporal and clinical diversity among patient samples analyzed. Significantly more XBB and less BA.5 variants were found after bivalent vaccination infection compared to other contemporaneous variants among those not receiving bivalent vaccine. Disclosures M. Carmen Frias-Kletecka, MD, Sanofi: Honoraria

Abstract We describe relapse of COVID-19 symptoms and SARS-CoV-2 viral load following nirmatrelvir/ritonavir (NM/R) in 8 non-immunocompromised patients aged 31 to 71-years-old. Most patients improved rapidly after treatment with NM/R and had negative antigen or PCR tests prior to relapse on Days 9-12 of their illness. Relapse symptoms were described most frequently as cold symptoms, though some patients experiencing a recurrence of fatigue and headache. All relapses resolved without additional antiviral treatment. Viral load during relapse was comparable to levels during initial infection. Sequencing in three patients indicated that relapse was not due to a treatment-emergent mutation or infection with a different viral strain. One patient transmitted SARS-CoV-2 to two family members during relapse. The presence of high viral load and the occurrence of one transmission event suggest that patients with relapse should isolate until antigen testing is negative.

BACKGROUND:The immunogenicities of polypeptide blood group antigens vary, despite most being created by single amino acid (AA) substitutions. To study the basis of these differences, we employed an immunoinformatics approach to determine whether AA substitution sites of blood group antigens have structural features typical of B-cell epitopes and whether the extent of B-cell epitope properties is positively related to immunogenicity.STUDY DESIGN AND METHODS:Fifteen structural property prediction programs were used to determine the likelihood of β-turns, surface accessibility, flexibility, hydrophilicity, particular AA composition and AA pairs, and other B-cell epitope properties at AA substitution sites of polypeptide blood group antigens.RESULTS:AA substitution sites of Lua , Jka , E, c, M, Fya , C, and S were each located in regions with at least two structural features typical of B-cell epitopes. The substitution site of K, the most immunogenic non-ABO/D antigen, scored the lowest for most B-cell epitope properties and was the only one not predicted to be part of a linear B-cell epitope. The most immunogenic antigens studied (K, Jka , Lua , E) had B-cell epitope structural properties determined by the fewest programs; the least immunogenic antigens (e.g., Fya , S, C, c) had B-cell epitope properties according to the most programs.DISCUSSION:Counter to prediction, the immunogenicity of polypeptide blood group antigens was not positively related to B-cell epitope structural features present at their AA-substitution sites. Instead, it tended to be negatively related. The AA-substitution site of the most immunogenic non-ABO/D antigen, K, had the least B-cell epitope features.

Rebound of SARS-CoV-2 Infection after Nirmatrelvir-Ritonavir TreatmentTo the Editor: Nirmatrelvir is an inhibitor of the main protease in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has been shown to block viral replication and reduce disease severity in unvaccinated persons at risk for the progression of coronavirus disease 2019 . 1 Here, we describe the occurrence of rebound symptoms and viral replication after treatment with nirmatrelvir combined with ritonavir.Patient 1, a 71-year-old man with asthma, reported having rhinorrhea, sore throat, congestion, cough, fatigue, malaise, chills, fever (temperature, 38.4°C), and a positive rapid antigen test for SARS-CoV-2.A 5-day course of nirmatrelvirritonavir was started on the same day.He was asymptomatic from day 2 through day 8. On days 9 through 12, while the patient was still isolating, he had a return of typical cold symptoms with rhinorrhea, sore throat, and congestion along with increased asthma symptoms.SARS-CoV-2 viral load was determined from anterior nasal swabs according to the cycle threshold (Ct) on quantitative reverse-transcriptasepolymerase-chain-reaction (RT-PCR) assay and indirectly from results of antigen testing.Peaks of symptoms and viral load coincided on days 1 and 9 (Fig. 1 and Fig. S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org).Whole-genome viral sequencing identified the BA.1.20subvariant of the SARS-CoV-2 B.1.1.529(omicron) variant from day 1 through day 11.Patient 2, a 69-year-old man, had cold symptoms and positive results on rapid antigen testing and PCR assay on day 0 through day 3.A 5-day course of nirmatrelvir-ritonavir was started on day 1.The patient was asymptomatic with negative results on rapid antigen testing and intermittent PCR assays from day 4 to day 9. Mild cold symptoms and positive results on both rapid antigen testing and RT-PCR assay recurred on day 10 and lasted for 3 days.Patient 3, a 50-year-old woman who lived in the same household with Patient 2, had a similar pattern of rebound symptoms and viral load after treatment with nirmatrelvir-ritonavir.Viral sequencing identified the omicron BA.2.9 subvariant in these two patients.In all three patients who have been described here, there were no muta-The New England Journal of Medicine Downloaded from nejm.org

We aimed to characterize clinical and demographic factors affecting clinical outcomes of COVID-19 and describe viral epidemiology among unvaccinated Veterans in New England. Veterans infected with COVID-19 in Veterans Administration healthcare systems in six New England states from April 8, 2020, to September 2, 2021, were correlated with outcomes of 30-day mortality, nonpsychiatric hospitalization, and intensive care unit admission (ICU-care). We sequenced 827 viral genomes. Of 3950 Veterans with COVID-19 before full vaccination, 81% were White, 8% were women, and the mean age was 60 years. Overall, 19% of Veterans required hospitalization, 2.8% required ICU care, and 4.9% died. In this largely male and older cohort, poor outcomes correlated with increasing age. Most New England Veterans (>97%) were infected with B.1 sublineages with the D614G mutation in 2020 and early 2021. B.1.617.2 lineage (68%) predominated after July 2021.

Abstract We describe relapse of COVID-19 symptoms and SARS-CoV-2 viral load following nirmatrelvir/ritonavir (NM/R) in 8 non-immunocompromised patients aged 31 to 71-years-old. Most patients improved rapidly after treatment with NM/R and had negative antigen or PCR tests prior to relapse on Days 9-12 of their illness. Relapse symptoms were described most frequently as cold symptoms, though some patients experiencing a recurrence of fatigue and headache. All relapses resolved without additional antiviral treatment. Viral load during relapse was comparable to levels during initial infection. Sequencing in three patients indicated that relapse was not due to a treatment-emergent mutation or infection with a different viral strain. One patient transmitted SARS-CoV-2 to two family members during relapse. The presence of high viral load and the occurrence of one transmission event suggest that patients with relapse should isolate until antigen testing is negative.

Abstract We describe relapse of COVID-19 symptoms and SARS-CoV-2 viral load following nirmatrelvir/ritonavir (NM/R) in 10 non-immunocompromised patients aged 31 to 71-years-old. Most patients improved rapidly after treatment with NM/R and had negative antigen or PCR tests prior to relapse on Days 9-12 of their illness. Relapse symptoms were described most frequently as cold symptoms, though some patients experiencing a recurrence of fatigue and headache. All relapses resolved without additional antiviral treatment. Viral load during relapse was comparable to levels during initial infection. Sequencing in three patients indicated that relapse was not due to a treatment-emergent mutation or infection with a different viral strain. One symptomatic and one presymptomatic patient transmitted SARS-CoV-2 to family members during relapse. The presence of high viral load and the occurrence of two transmission events suggest that patients with relapse should isolate until antigen testing is negative.

Abstract Initiation of NM/R treatment on Day 0 in a 71-year-old vaccinated and boosted male resulted in rapid resolution of COVID-19 symptoms followed one week later by the development of typical cold symptoms. SARS-CoV-2 viral load fluctuated in parallel with symptoms, with two distinct peaks on Day 1 and Day 9 of illness. No other respiratory pathogens were identified. Viral samples demonstrated sequence identity for the omicron subvariant BA.1 on Days 1, 7, and 11. Our findings suggest that viral replication and COVID-19 symptoms may recur after very early treatment with NM/R before natural immunity is sufficient to fully clear SARS-CoV-2.

See article on page 2255–2264, in this issue

Background The global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown. Methods We compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay. Results Of the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%). Conclusion We found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.

1Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD, United States of America; 2Jiangsu Province Blood Center, Nanjing, China; 3University of Campinas, Campinas, Brazil; 4American Red Cross Biomedical Services, Philadelphia, PA, United States of America; 5Cedars Sinai Medical Center, Los Angeles, CA, United States of America; 6Brigham and Women's Hospital, Boston, MA, United States of America; 7EFS Île-de-France, Creteil, France; 8Yale University School of Medicine, New Haven, CT, United States of America; 9Héma-Québec, Québec, Québec, Canada; 10Indiana Blood Center, Indianapolis, IN, United States of America; 11German Red Cross Blood Service Baden-Wurttemberg-Hessen, Ulm, Germany; 12Hospital Sirio Libanês, São Paulo, Brazil; 13Blood Center of Wisconsin, Milwaukee, WI, United States of America

BACKGROUNDThe intrinsic properties of polypeptide blood group antigens that determine their relative immunogenicities are unknown. Because size, composition, charge, dose, and epitope glycosylation affect the immunogenicity of other polypeptides, we examined whether similar properties were related to the immunogenicity of blood group antigens. STUDY DESIGN AND METHODSAmino acid (AA) sequences of antithetical blood group antigens were searched for N- and O-glycosylation sites. Regression analysis was carried out to determine whether blood group protein properties, including total and ectodomain size, red blood cell (RBC) antigen site density, number of mismatched AAs between an antigen and its closest homolog, and differences in mass, charge, and hydrophobicity of the mismatched AAs, were related to immunogenicity. RESULTSThe immunogenicities of non-RhD polypeptide antigens were directly related to the total and ectodomain sizes of their carrier proteins. A negative power relationship existed between RBC antigen site density and immunogenicity, such that the most immunogenic antigens had the lowest site density. The strong immunogenicity of RhD was related to the number of AA mismatches between RhD and RhCE, to their cumulative hydrophobicity and electrostatic mismatch scores, and the cumulative AA mass difference. No N- or O-glycosylation differences were predicted for antithetical or homologous antigens, other than a previously known N-glycosylation difference between K and k. CONCLUSIONEpitope glycosylation appeared not to be a determinant of immunogenicity for blood group antigens, except possibly for K. The immunogenicity of blood group antigens was positively related to total and ectodomain sizes of blood group proteins and negatively related to antigen site density. Such findings should be considered hypothesis generating for future, more definitive studies.

BACKGROUNDThe immunogenicities of polypeptide blood group antigens vary widely. One possible determinant of immunogenicity is antigenic foreignness. The goal was to employ alternative ways of assessing foreignness and determine whether foreignness was related to immunogenicity.STUDY DESIGN AND METHODSForeignness was assessed as the extent of protein functional disruption caused by the exofacial amino acid (AA) substitutions that create blood group antigens, using AA substitution prediction algorithms such as Meta‐SNP and according to whether those substitutions were radical or conservative.RESULTSAA substitutions that create the most immunogenic antigens had the highest Meta‐SNP scores, predictive of greater protein structure and function changes. Four of the 11 exofacial AAs that distinguish the most immunogenic antigen, RhD, from RhCE, and substitutions creating four of the five next most immunogenic antigens had the highest Meta‐SNP scores (0.293‐0.649). Excluding the outlier Jka, the mean Meta‐SNP score of the four most immunogenic non‐RhD antigens (K, Lua, E, c) was 3.7‐fold higher than the mean of the four least immunogenic (M, Fya, C, S), 0.459 versus 0.123 (p = 0.0026). Regression analysis revealed a relationship between immunogenicity and Meta‐SNP score (R2 = 0.953). Actual protein functional disruption was predicted for the AA substitution creating the E antigen. An AA cluster at Positions 350, 353, and 354 of RhD was unique, containing radical substitutions according to two classification schemes and relatively high Meta‐SNP scores (0.351‐0.432).CONCLUSIONThe immunogenicity of blood group antigens was related to the functional disruption caused by the AA substitutions that create the antigens, as measured by Meta‐SNP score.

BACKGROUND Failure to detect non‐ABO blood group alloantibodies places patients at risk for hemolytic reactions. Suboptimal alloantibody detection could result from posttransfusion testing performed too early, too late, or not at all. Testing performed too early may precede antibody induction, while testing performed too late could miss antibodies that have evanesced. Taking these factors into account, our goal was to determine the percentage of alloantibodies detected with real‐world testing practices. STUDY DESIGN AND METHODS The alloantibody detection rate in a general hospital setting was determined based on the frequency and timing of antibody testing after red blood cell (RBC) transfusions and rates of antibody induction and evanescence. Intervals to follow up testing after RBC transfusions (n = 561 RBC units in 100 random patients) were determined retrospectively. Best‐fit lines and equations for antibody induction and evanescence were computed on previously published data. RESULTS Nearly half (271/561; 48.3%) of RBC infusions had either no follow‐up antibody screen or testing too soon (<30 days) after transfusion to detect alloimmunization. Of the remaining RBC units, 10.3% (58/561) had follow‐up testing 30 to 112 days posttransfusion, 28.7% (161/561) were followed up at more than 112 days, and 12.7% (71/561) were tested at both 30 to 112 days and more than 112 days. By inputting these timing data into best‐fit line equations for antibody induction and evanescence, we calculated an alloantibody detection rate of 31.6%. CONCLUSION Posttransfusion antibody testing was inadequately timed for optimal alloantibody detection. Real‐world compatibility testing was predicted to detect less than one‐third of non‐ABO antibodies, thereby exposing patients to risks of mismatched transfusion.