Prevalence and duration of anti-SARS-CoV-2 antibodies in healthcare workers

Introduction. Knowledge of the seroprevalence and duration of antibodies against SARS-CoV-2 was needed in the early phases of the COVID-19 pandemic and is still necessary for policy makers and healthcare professionals. This information allows us to better understand the risk of reinfection in previously infected individuals. Methods. We investigated the prevalence and duration of detectable antibodies against SARS-CoV-2 in sequentially collected samples from 379 healthcare professionals. Results. SARS-CoV-2 seroprevalence at inclusion was 5.3% (95% confidence interval (CI): 3.3-8.0%) and 25% of seropositive participants reverted during follow-up. At the end of follow-up, the calculated probability of having detectable antibodies among former seropositive participants was 72.2% (95% CI: 54.2-96.2%). Conclusion. Antibodies against SARS-CoV-2 were detectable in a subset of infected individuals for a minimum of 39 weeks.

Prospective Evaluation of Serial Protein Biomarker Measurements in Sepsis-Induced Acute Respiratory Distress Syndrome

RATIONALE: Protein biomarkers including soluble receptor for advanced glycation end-products (sRAGE), angiopoietin-2 (Ang- 2), and surfactant protein-D (SP-D) have been studied for diagnosis or prognostication in acute respiratory distress syndrome (ARDS). However, prior studies of ARDS biomarkers often included heterogeneous populations, and rarely examined serial measurements. Our aim was to determine the association between serially measured sRAGE, Ang-2, and SP-D levels and ARDS development in patients with sepsis. METHODS: Adult patients admitted to the medical ICU at Grady Memorial Hospital within 72 hours of sepsis diagnosis were enrolled into this prospective observational cohort study within 48 hours of ICU admission. Patients who already had ARDS at the time of screening were excluded. After obtaining informed consent, serial plasma samples were collected on day 1, 2, and 3 of enrollment, and were analyzed for sRAGE, Ang-2, and SP-D levels using ELISA. The patients were followed for up to 28 days for relevant clinical characteristics and outcomes. The primary outcome was ARDS development according to the Berlin Definition. The secondary outcome was mortality. Pulmonary sepsis was defined as the primary infection being pneumonia (including COVID19) or aspiration pneumonia. The biomarker levels and their changes from day 1 to days 2/3 were compared between those who developed ARDS versus those who did not. RESULTS: Among 80 patients with sepsis enrolled between September 1, 2020 and June 22, 2021, 15 patients (18.8%) developed ARDS and 65 patients (81.3%) did not. ARDS patients had higher proportions of pulmonary sepsis (14/15 [93.3%] vs. 30/65 [46.2%], p=0.001) and COVID19 (7/15 [46.7%] vs. 7/65 [10.8%], p=0.003) compared to non-ARDS patients. ARDS patients had higher SP-D levels on days 1 and 2, and had a greater increase in sRAGE levels from day 1 to day 3, compared to non-ARDS patients (Figure 1A- 1B). Within the ARDS group, those who died had higher sRAGE levels on day 1 compared to those who survived (Figure 1C). CONCLUSIONS: In this analysis, ARDS patients had higher SP-D and a greater increase in sRAGE over time compared to non- ARDS patients. Non-survivors of ARDS also had higher sRAGE compared to survivors. Our findings suggest that early serial biomarker measurements may be useful for identifying sepsis patients at risk of developing ARDS and adverse clinical outcomes, and for risk stratifying sepsis patients in ARDS clinical trials focused on early therapeutics and prevention. Larger studies are needed for more detailed analyses and confirmation of these findings.

Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Background. Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC). Methods. A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y (Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Figure. Spike SNP distinguishes mutations occurring in different lineages (A-C). Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type);pink B.1.1.7;purple, B1.525;and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D). Results. The lower limit of 95% detection was 2.46 to 2.48 log10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNAmodified probes. Conclusion. The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally.