SARS-CoV-2 ANTIBODY RESPONSES POST-INFECTION IN PREGNANCY BY VACCINATION STATUS

Background: We evaluated SARS-CoV-2 antibody binding and neutralization responses at delivery among pregnant persons with prior SARS-CoV-2 infection by vaccine status. Method(s): We enrolled participants with evidence of prior SARS-CoV-2 infection detected in pregnancy (anti-nucleocapsid [anti-N] IgG+ on enrollment or prior RT-PCR+ or antigen+) and followed them through delivery. Maternal delivery and cord blood samples were tested for SARS-CoV-2 binding antibodies to spike (anti-S) (from vaccination and/or infection) and anti-N (from infection only) IgG by Abbott Architect followed by neutralizing antibodies (classified as neutralizing if serum dilution inhibited infection by 50% [ND50 heat] >=20 and R2 >=0.9) if sample volume allowed. Positive IgG thresholds were Abbott index >=1.4 for anti-N and >=50 AU/mL for anti-S. Chi-squared test was used to compare differences in proportions between groups. Wilcoxon rank sum test was used to compare medians. Result(s): Among 71 participants with delivery and cord samples, median age was 33 years (interquartile range [IQR] 30-35) and median gestational age was 31.7 weeks (IQR 18.0-37.9) at enrollment in pregnancy. By delivery, 17 (24%) participants were unvaccinated, 21 (30%) were partially vaccinated or had completed a primary series, and 33 (46%) were boosted. Median time from infection (RT-PCR+ or antigen+ result) to delivery was 16.7 weeks (IQR 9.7- 24.3). At delivery, 33 (46%) of maternal (median 3.2 index) and 37 (52%) of cord samples (median 3.1 index) were anti-N IgG+. Participants with >=1 vaccine were more likely to be anti-S IgG+ than those unvaccinated (100% vs. 82%, p< 0.01), have higher median anti-S IgG+ (25,000 vs 1,019 AU/ml, p< 0.01), and have neutralizing antibodies (100% vs. 81%, p< 0.01) with higher median log10 neutralization (1:4.00 vs 1:2.41, p< 0.01) at delivery. Similarly, cord blood from participants with >=1 vaccine was more likely to be anti-S IgG+ than those unvaccinated (100% vs. 82%, p< 0.01), have higher median anti-S IgG+ (25,000 vs 1,188 AU/ml, p< 0.01), and have neutralizing antibodies (100% vs. 75%, p< 0.01) with higher median log10 neutralization (1:4.00 vs 1:2.41, p< 0.01) at delivery. Conclusion(s): Among pregnant people with prior SARS-CoV-2 infection detected during pregnancy, maternal and cord blood antibody binding and neutralization responses were higher among those receiving SARS-CoV-2 vaccination prior to delivery. (Table Presented).

Symptom and Viral Rebound in Untreated Covid-19 Infection

Background: Rebound of SARS-CoV-2 RNA and symptoms has been reported in people treated with nirmatrelvir/ritonavir. Since the natural course of viral and symptom trajectories during COVID-19 have not been well described, we evaluated the incidence of viral rebound and symptom relapse in untreated individuals with mild-to-moderate COVID-19. Method(s): This analysis included 563 participants randomized to placebo in the ACTIV-2/A5401 platform trial. Participants recorded the severity (scored as 0-3) of each of 13 targeted symptoms daily from days 0-28, with symptom score being the summed score (0-39). Symptom rebound was defined as >=4 point increase in symptom score between the maximum and the preceding minimum score. Anterior nasal (AN) swabs were collected for SARS-CoV-2 RNA testing on days 0-14 and 28. Viral rebound was defined as a >=0.5 log10 RNA copies/mL increase from the immediately preceding time point to a level >=3.0 log10 RNA copies/mL, with high-level rebound defined as an increase of >=0.5 log10 copies/mL to a level >=5.0 log10 RNA copies/mL. To mirror the timing of a 5-day nirmatrelvir/ritonavir course, a supportive analysis was conducted where participants were only classified as rebounders if their rebounds occurred on or after day 5. Result(s): Symptom rebound was identified in 26% of participants at a median [Q1, Q3] of 6 [4, 9] days after study entry and 11 [9, 14] days after initial symptom onset. Individuals with symptom rebound were more likely to be female, at high risk for progression to severe disease, have shorter time since symptom onset at study entry, and have higher symptom score and higher AN viral levels day 0. Viral rebound was detected in 32%, with high-level rebound in 13% of participants. Participants with viral rebound were older, more likely to be at low risk for progression to severe disease and had higher median AN viral level at day 0. Most symptom and viral rebound were transient with 89% of symptom rebound and 95% of viral rebound events occurring for only a single day before improving. The combination of symptom and high-level viral rebound was observed in 3% of participants. In the supportive analysis of rebound occurring >=5 days after study entry, 22% and 20% of participants met symptom and viral rebound criteria, respectively, but only 1.2% of participants met criteria for both symptom and high-level viral rebound. Conclusion(s): Symptom or viral rebound in the absence of antiviral treatment is common, but the combination of symptom and viral rebound is rare.

Longitudinal SARS-COV-2 anti-spike antibody response in pregnant people with natural infection and variable vaccine uptake

Background. Natural SARS-CoV-2 infection results in anti-nucleocapsid (N) and anti-spike (S) antibody (Ab) development. Anti-S Ab response (conferred by infection and/or vaccination) is more closely associated with protection. We evaluated anti-N/S Ab responses in vaccinated (> 1 dose) and unvaccinated pregnant people with prior SAR-CoV-2 infection. Methods. During January 2021-March 2022, we enrolled participants with SARS-CoV-2 infection identified in pregnancy (26 via anti-N IgG+;52 via prior RT-PCR+). Baseline, 1, 2, 3, 6, and 12 months, and delivery samples were tested for anti-N (index >= 1.4 positive) and anti-S (>= 50 AU/mL positive) IgG Ab by Abbott Architect. Kaplan-Meier methods were used to measure Ab response duration. Results. Among 78 participants, 62 (79%) enrolled in pregnancy (median 27 weeks gestation), and 16 (21%) at delivery/postpartum (median 2 weeks);34 (44%) had received >=1 vaccine prior to initial Ab testing. At baseline, 59 (75%) participants had concordant anti-N/S positive results (median anti-N index 3.58 [IQR 2.01-5.82], median anti-S 5529 AU/ml [IQR 687-25000]). Anti-S IgG was higher (25000 vs 774, p< 0.001) among participants receiving >=1 vaccine vs no vaccine, while anti-N IgG indices were similar. Among 59 participants with initial anti-N IgG+ results, median time to anti-N IgG negative results was 31 weeks after first RT-PCR+ (median 17 weeks after first anti-N IgG+ result). Only 1 (unvaccinated) participant had an anti-S IgG negative result by 22 weeks after first RT-PCR+ result. Among 30 participants with delivery samples (median 16 weeks after RT-PCR+, 12 weeks after baseline anti-N IgG+ samples), 15 (52%) remained anti-N IgG+;29 (97%) remained anti-S IgG+. Anti-S IgG was higher (25000 vs 826 AU/ml, p< 0.001) among participants receiving >= 1 vaccine vs. no vaccine prior to delivery. Conclusion. Among pregnant persons with prior SARS-CoV-2 infection, duration of anti-S IgG response was longer than anti-N IgG irrespective of vaccine status;vaccination during pregnancy was associated with higher anti-S levels at baseline and delivery. While anti-S IgG were detectable for >= 6 months, longer term follow-up is needed to assess durability of hybrid immunity vs. infection alone and has implications for maternal and infant protection.

Evaluation of a heterologous booster vaccine regimen: Pfizer-BioNTech BNT162b2 mRNA booster vaccine following priming with Novavax NVX-CoV2373

Background. In the United States, booster vaccines for persons 18 years and older were approved under Emergency Use Authorization (EUA) in September 2021. Waning immunity following SARS-CoV-2 primary vaccination series led to recommendations for booster vaccination. Emerging data suggest that providing boosters different from the primary series (heterologous vaccination) may provide a broader immune response than boosting with the same vaccine (homologous vaccination). CDC recommended the Pfizer-BioNTech BNT162b2 30-mug mRNA booster vaccine to clinical trial participants >6 months post study vaccines if not planned for boosting within the study. Methods. We conducted an observational study of persons who received 2 doses of Novavax protein-based NVX-CoV2373 vaccine 21 days apart, in a Phase 3 clinical trial, and subsequently received a Pfizer BNT162b2 booster vaccine under EUA. Serologic assays, including the Roche anti-nucleocapsid (N) IgG and anti-Spike (S) IgG, were performed on blood collected pre-booster (D0) and on days 18 (D18) and 34 (D34) post-booster vaccine. The anti-S IgG geometric means (GMTs) were calculated over study time points. Wilcoxon signed rank test was performed to compare anti-S IgG response between D0 and D18 and D0 and D34. Results. Of 26 participants enrolled, 16 (57%) were women;the median age was 47 years (range 29-67). Roche anti-N antibodies were negative at all visits. Time from second NVX-CoV2373 vaccine to Pfizer BNT162b2 booster was a median of 10.4 months in 54% of participants and 7 months in 46% of participants. Anti-S IgG GMTs were 222 BAU/ml D0, 24,723 BAU/ml D18, and 24,584 BAU/ml D34 (p< 0.0001 for comparisons of D0 with D18 & D34). Overall, participants tolerated the booster vaccine without significant adverse events. Cell mediated immunity and D614G pseudovirus neutralizing antibody assays are in progress. Figure 1. Anti-S IgG titers pre and post-booster vaccine 16 participants included with all 3-time study time points for comparison. Conclusion. Two doses of NVX-CoV2373 vaccine followed by the Pfizer BNT162b2 booster vaccine resulted in ~100-fold increase in anti-S IgG against SARS-CoV-2. No participant had evidence of prior SARS-CoV-2 infection by anti-N IgG. Two doses of NVX-CoV2373 vaccine followed by one dose of Pfizer BNT162b2 vaccine is an effective and well-tolerated regimen for boosting anti-S IgG against SARS-CoV-2.

Resistance Analyses of Patient Viral Samples from the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1)

Background. Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in non-hospitalized and hospitalized adult as well as pediatric patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 ACTT-1 placebo-controlled clinical trial in hospitalized adults. Methods. Oro- or nasopharyngeal swab samples in ACTT-1 study were collected on Day 1, 3, 5, 8, 11, 15, and 29. All participants with >80th and 50% of participants with < 20th percentile of cumulative viral shedding underwent resistance analysis in both the RDV and placebo arm. The SARS-CoV-2 genome was sequenced using next generation sequencing. Phenotyping was conducted using virus isolation from clinical samples or generation of select site-directed mutants (SDMs) in a SARS-CoV-2 replicon system. Results. The majority of the sequencing data were obtained from participants with 80th percentile of cumulative viral shedding from the RDV and placebo arms as shown in Table 1. Among participants with both baseline and postbaseline sequencing data, emergent substitutions in nsp12 were observed in 12 of 31 participants (38.7%) treated with RDV and 12 of 30 participants (40.0%) in the placebo arm. The nsp12 substitutions that emerged in the RDV arm were only observed in one participant each, and the majority were present as mixtures with wildtype sequence. The following nsp12 mutations emerged in the RDV treatment group and were successfully phenotyped as clinical isolates or SDMs with low to no fold change in RDV susceptibility: A16V (0.8-fold), P323L+V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N+V792I (3.4-fold). V792I and C799F were identified previously in vitro in resistance selection experiments (Stevens Sci Transl Med 2022). In addition, for D684N and V764L identified in the RDV arm, the recovery of neither clinical isolates nor SDMs for phenotypic analysis were successful. Conclusion. The similar rate of emerging nsp12 substitutions in participants treated with RDV compared to placebo and the minimal to no change in RDV susceptibility among the treatment-emergent nsp12 substitutions support a high barrier to RDV resistance development in COVID-19 patients.

Diagnostic Test Kinetics, Infectivity, and Immunological Responses Among Unvaccinated Adults During Acute SARS-CoV-2 Infection

Background. Appropriate diagnostic testing can be used to inform infection control measures and reduce SARS-CoV-2 transmission, yet the test kinetics, infectivity, and immunological responses during acute, non-severe SARS-CoV-2 infection need clarity. Methods. We conducted a prospective cohort study between Nov 2020-July 2021 in Seattle, Washington of 95 unvaccinated, immunocompetent adults with no prior SARS-CoV-2 infection. Nasal swabs (nasopharyngeal and anterior) and blood serum samples were serially collected at six visits over two months. Viral RNA, N and S antigen concentrations, and viral growth/infectivity were measured from nasal samples. Anti-S total antibody and IgG assays were performed on serum. We fit loess curves to quantitative data corresponding to each testing modality by days since symptom onset (DSSO) and compared qualitative test results across time points to demonstrate timedependent agreement of PCR, N antigen, and culture results. Generalized estimating equations were used to approximate relative risk of culture positivity (a proxy for infectiousness) for positive vs. negative test results (antigen and PCR), stratified by presence/ absence of symptoms and DSSO. Sampling Schema Nasal swabs and venous blood were collected at visits 1-4;venous blood only at visits 5-6. All participants were enrolled within 14 days of symptom onset (median: 6) and 7 days of a positive test (median: 4). Results. Infections in this cohort (median age: 29y) were mild (no hospitalization). Median (IQR) time to negative result was 11 (4), 13 (6), and 20 (7) DSSO for culture growth, N antigen, and PCR tests, respectively. Viral RNA quantities declined more slowly than antigen and culturable virus;antibody titers rose rapidly 5-15 DSSO and plateaued 20-30 DSSO. All culture-positive samples collected 0-5 DSSO were positive by PCR, but relative risk of culture positivity (infectiousness) for positive vs. negative PCR results declined 6-10 DSSO. Relative risk of culture positivity for positive vs. negative antigen results was consistently high 0-10 DSSO, with similar results when stratified by presence of symptoms. Diagnostic test kinetics and immunological responses Diagnostic test kinetics and immunological responses measured in adults with non-severe, symptomatic SARS-CoV-2 infection: loess trendlines and 95% confidence intervals are given for SARS-CoV-2 viral load (calculated from PCR Ct value using a calibration curve), TCID50 from viral culture, mean concentrations of nucleocapsid and spike antigen proteins, and anti-S total and IgG antibody concentrations. Conclusion. The results reinforce the importance of molecular PCR testing as a highly sensitive diagnostic tool but with limited utility as an indicator of viral culturability and likely infectiousness. N antigen testing may be a preferable diagnostic test within two weeks of symptom onset, especially 6-10 DSSO, because it more closely correlates with culture growth over the course of infection.

Viral and Immunologic biomarkers of COVID-19 improve risk stratification and identify patients most likely to benefit from therapy with remdesivir

Background. ACTT-1 demonstrated clinical efficacy of remdesivir (RDV) in hospitalized patients with COVID-19;subgroup analyses suggested those most likely to benefit presented with milder clinical illness. To further clarify what subsets of hospitalized patients might benefit from RDV, we analyzed virological and immunological biomarkers in this previously reported cohort. Methods. Serum and upper respiratory tract (URT) swabs were collected on Day 1, 3, 5, 8, and 11 while hospitalized;Day 15 and 29 as able were collected and tested for quantitative RNA (URT and plasma), serum nucleoprotein (NPR), IL-6, CRP through Day 6, and serostatus (baseline only). Participants with a baseline and at least one subsequent sample were used in this analysis. Associations of all these biomarkers with clinical outcomes (mortality, recovery) and response to therapy were assessed. Of the 1062 participants in ACTT-1, 642 had baseline and at least one subsequent sample within 6 days of randomization (Fig 1, Table 1). Results. RDV-treated patients with moderate/severe disease who had elevated baseline NPR levels recovered faster (RRR 1.95 vs 1.04, p = 0.01);similar trends were noted for plasma and URT RNA levels (Fig 2A);mortality treatment effects by viral load subgroups (high or low) were not seen (Fig 2B). In patients with less severe illness, RDV treatment was associated with an accelerated decline in NPR (difference -0.062 log10 pg/ml per day, p = 0.003) and plasma RNA levels (difference -0.040 log10 pg/ml per day, p = 0.004. Fig 3A), and a decrease in the proportion of patients with increasing and/or persistent viral loads (Fig 3B). Patients with increasing/persistent viral loads also took longer to recover than those with decreasing viral loads, irrespective of disease severity: RRR for plasma RNA 0.45, 95% CI 0.28-0.73, RRR for NPR 0.44, 95% CI 0.22-0.88 for moderate/severe disease;RRR for plasma RNA 0.26, 95% CI 0.10 - 0.70 , RRR for NPR n.e. (no recoveries) for critical disease (Fig 4). Conclusion. Our study demonstrates a systemic antiviral effect of remdesivir, shows the prognostic value of viral and immunologic biomarkers for mortality and failure to recover, and identifies a group of hospitalized patients with COVID-19 most likely to benefit from remdesivir treatment. (Figure Presented).

Case Study: Impact of Diurnal Variations and Stormwater Dilution on SARS-CoV-2 RNA Signal Intensity at Neighborhood Scale Wastewater Pumping Stations

Wastewater based epidemiology (WBE) has emerged as a tool to track the spread of SARS-CoV-2. However, sampling at wastewater treatment plants (WWTPs) cannot identify transmission hotspots within a city. Here, we sought to understand the diurnal variations (24 h) in SARS-CoV-2 RNA titers at the m A neighborhood level, using pump stations that serve vulnerable communities (e.g., essential workers, more diverse communities). Hourly composite samples were collected from wastewater pump stations located in (i) a residential area and (ii) a shopping district. In the residential area, SARS-CoV-2 RNA concentration (N1, N2, and E assays) varied by up to 42-fold within a 24 h period. The highest viral load was observed between 5 and 7 am, when viral RNA was not diluted by stormwater. Normalizing peak concentrations during this time window with nutrient concentrations (N and P) enabled correcting for rainfall to connect sewage to clinical cases reported in the sewershed. Data from the shopping district pump station were inconsistent, probably due to the fluctuation of customers shopping at the mall. This work indicates pump stations serving the residential area offer a narrow time period of high signal intensity that could improve the sensitivity of WBE, and tracer compounds (N, P concentration) can be used to normalize SARS-CoV-2 signals during rainfall.

Oral ixazomib in untreated follicular lymphoma permits COVID-19 vaccine response and its efficacy is associated with clinical factors and gene expression signatures

Background: Standard chemoimmunotherapy for first-line treatment of follicular lymphoma (FL) achieves high rates of disease control but is not curative and carries significant toxicities including prolonged immunosuppression that may attenuate response to vaccinations (Marcus et al., NEJM 2017). While proteasome inhibitors have shown modest activity in R/R FL (Goy et al., JCO 2005), limited data address their use frontline. The comparatively favorable toxicity profile and convenient oral dosing of ixazomib support its investigation in this space. Methods: We evaluated ixazomib and its combination with short-course rituximab (R) for FL as part of an open-label, phase II investigator-initiated trial at the University of Washington / Fred Hutch Cancer Research Center / Seattle Cancer Care Alliance (NCT 02339922). Eligibility included an indication for treatment per NCCN guidelines and no prior standard systemic FL therapy. Ixazomib was administered at 4 mg orally once a week until disease progression or unmanageable toxicity. One course of R at 4 weekly doses of 375 mg/m2 was added during the 7th 28-day cycle, after an initial 6-cycle “window” on ixazomib alone. Available pretreatment formalin-fixed, paraffin-embedded tissue biopsies were subjected to RNA extraction by standard methods and gene expression profiling (GEP) using the NanoString™ PanCancer IO 360 panel to query pathways in proteasomal degradation and lymphomagenesis. Standard GEP quality control and data processing were performed with the ROSALIND® platform. Patients vaccinated per standard of care for COVID-19 while actively receiving ixazomib and ≥ 6 mo after completing R were evaluated for serologic response ≥ 2 weeks after the final dose of vaccine using the Roche Elecsys® Anti-SARS-CoV-2 S assay against the spike protein receptor binding domain. Results: Twenty pts began therapy between Feb 2017 and January 2020. All had grade I/II FL and FLIPI score was 2 in 20% and ≥ 3 in 20%;FLIPI score in all other patients was 0 or 1. Eleven (55%) pts met GELF criteria for high tumor burden disease including 6 (30%) pts with a tumor mass ≥ 7 cm. Median follow-up was 32.1 months (range 5.7 - 51.6). The ORR by Lugano criteria was 35% (CR 5%) during the ixazomib window and 65% (CR 45%) overall. At data cut (June 15, 2021) all patients were alive and 8 (40%) remained progression-free on treatment (Figure 1). By KM estimate, median PFS was 25.8 mo and median DOR was not reached at a median follow-up of 29.6 mo. As expected, high-grade treatment-related AEs were infrequent for ixazomib and R, including grade ≥ 3 events in 3 unique pts (15%;diarrhea, transaminitis, and cytopenias). No grade ≥ 4 or serious AEs were observed. Toxicities led to study-directed drug interruptions in 4 (20%) pts and dose reduction to ixazomib 3 mg weekly in 2 pts (10%). Higher ORR to ixazomib monotherapy was associated with FLIPI > 1 (p = 0.04) and, by exploratory GEP, downregulation of components of proteasomal degradation and upregulation of NF-KB and chemokine signaling (Figure 2). High tumor burden by GELF (p = 0.89) and tumor mass ≥ 7 cm (p = 0.26) were not associated with ORR to ixazomib. All 6 of 6 patients evaluated to date for response to COVID-19 vaccination, administered at a median of 32.5 mo (range 7.0 - 41.0) after last dose of R, achieved positive anti-spike protein antibodies (median anti-S 163.8 AU/mL, range 13.3 - 1139);none was diagnosed with COVID-19. Conclusions: The simple outpatient regimen of weekly oral ixazomib and the addition of 4 doses of R shows significant long-term activity with low toxicity in untreated FL. Extended DOR is achievable especially in patients who respond to ixazomib monotherapy. Ixazomib efficacy was associated with higher FLIPI scores and gene expression signatures implicated in proteasomal degradation and B-cell signaling pathways. Ixazomib deserves further investigation as a biomarker-driven therapeutic in untreated FL, particularly as an option that prioritizes outpatient management and serologic responsiveness to im unization. (Figure Presented).

SARS-CoV-2 Seroprevalence and Antibody Response among Pregnant People in Seattle, WA

Background. Antenatal care is a unique opportunity to assess SARS-CoV-2 seroprevalence and antibody response in pregnant people, including those with previously unknown infection. Methods. Pregnant people were screened for SARS-CoV-2 IgG during antenatal care or delivery in Seattle, Washington with Abbott Architect chemiluminescent immunoassay which provides quantitative index (positive ≥1.4). Participants with IgG+ results or identified with RT-PCR+ results via medical records were invited to enroll in a longitudinal evaluation of antibody responses. We report preliminary results of an ongoing seroprevalence and longitudinal study with planned 18-month follow-up. Results. Between September 9, 2020-May 7, 2021, we screened 1304 pregnant people;62 (4.8%) tested SARS-CoV-2 IgG+, including 28 (45%) with known prior SARS-CoV-2 infection. Among participants testing IgG+, median age was 32 years (interquartile range [IQR] 26-35) and median gestational age was 21 weeks (IQR 12-38) at screening;median IgG index was 3.2 (IQR 2.1-4.9, range 1.4-9.9), including 3.9 (IQR 2.3-5.8) among those with vs. 2.7 (IQR 1.9-4.2) among those without prior RT-PCR+ results (p=0.05 by Wilcoxon rank-sum). Of 30 longitudinal study participants enrolled, 24 tested IgG+ at baseline (75% with prior RT-PCR+ result) and 6 tested IgG- on enrollment but were identified as previously RT-PCR+ via medical records;24/30 (80%) reported previous symptoms. Of 24 participants testing IgG+ at baseline, 14 (58%) had first follow-up IgG results at median of 66 days (IQR 42-104) since initial testing, with median IgG index of 2.0 (IQR 1.0-3.8). 9/14 (64%) participants with repeat IgG testing remained IgG+ at first follow-up (≤280 days after first RT-PCR+ result for those with and ≥104 days after first IgG detection for those without prior RT-PCR+ results), while 5/14 (26%) had a negative Abbott IgG test at a median of 81 days (IQR 75-112) since initial testing. Conclusion. Nearly half of pregnant people testing SARS-CoV-2 IgG+ reported no known prior SARS-CoV-2 diagnosis or symptoms. SARS-CoV-2 IgG antibody response and durability in pregnancy has implications for maternal and neonatal protection and susceptibility and highlights potential benefits of vaccination in this population.

Stability of SARS-CoV-2 in phosphate-buffered saline for molecular detection. (Special Issue: Diagnostic testing for severe acute respiratory syndrome coronavirus 2 and lessons from this pandemic.)

This study evaluated the stability of differing viral loads of SARS-CoV-2 over 28 days stored at room temperature, 4 degrees Celsius, -20 Celsius, or -80 Celsius. For the high concentration of SARS-CoV-2, regardless of storage conditions, 100% of samples were detected by qRT-PCR through day 28. At room temperature, median cycle threshold (CT) values for lower titers for both N1 and N2 targets remained consistent through day 28, fluctuating less than 1 median CT. For lower concentrations of virus, storage at room temperature was associated with reductions of positivity beginning at day 7, and by day 28, 0% of samples were detected for N1. Storage at room temperature was the least stable of all environmental conditions tested, with 54.2% of negative PCR results. At 4 degrees Celsius, there was minimal change in CTs over time at the higher viral concentration. For lower titers, CTs increased by 2.1 CTs for N1 and 2.6 CTs for N2 over the 28 days. At -20 degrees Celsius, lower titers of virus fluctuated slightly more, increasing by 3 CTs. Storage of SARS-CoV-2 in PBS at -20 degrees Celsius was the second least stable condition, accounting for 37.5% of negative PCR results. Storage at -80 degrees Celsius showed the greatest stability, with all samples detected throughout the 28 days and 1.5 median CTs for both N1 and N2 targets. Here, this study shows that the stability of SARS-CoV-2 can be maintained at 4 degrees Celsius for up to a month when -80 degrees Celsius storage is not available. At viral loads of >5,000 copies/ml corresponding to >75% of positive samples recovered in the clinical lab to date-different storage temperatures did not have a substantial impact on the ability to detect SARS-CoV-2 when stored in PBS.