Diminished Neutralization Capacity of SARS-CoV-2 Omicron BA.1 in Donor Plasma Collected from January to March 2021.

The 50% plaque reduction neutralization assay (PRNT50) has been previously used to assess the neutralization capacity of donor plasma against wild-type and variant of concern (VOC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging data suggest that plasma with an anti-SARS-CoV-2 level of ≥2 × 104 binding antibody units/mL (BAU/mL) protects against SARS-CoV-2 Omicron BA.1 infection. Specimens were collected using a cross-sectional random sampling approach. For PRNT50 studies, 63 previously analyzed specimens by PRNT50 versus SARS-CoV-2 wild-type, Alpha, Beta, Gamma, and Delta were analyzed by PRNT50 versus Omicron BA.1. The 63 specimens plus 4,390 specimens (randomly sampled regardless of serological evidence of infection) were also tested using the Abbott SARS-CoV-2 IgG II Quant assay (anti-spike [S]; Abbott, Chicago, IL, USA; Abbott Quant assay). In the vaccinated group, the percentages of specimens with any measurable PRNT50 versus wild-type or VOC were wild type (21/25 [84%]), Alpha (19/25 [76%]), Beta (18/25 [72%]), Gamma (13/25 [52%]), Delta (19/25 [76%]), and Omicron BA.1 (9/25 [36%]). In the unvaccinated group, the percentages of specimens with any measurable PRNT50 versus wild type or VOC were wild-type SARS-CoV-2 (16/39 [41%]), Alpha (16/39 [41%]), Beta (10/39 [26%]), Gamma (9/39 [23%]), Delta (16/39 [41%]), and Omicron BA.1 (0/39) (Fisher's exact tests, vaccinated versus unvaccinated for each variant, P < 0.05). None of the 4,453 specimens tested by the Abbott Quant assay had a binding capacity of ≥2 × 104 BAU/mL. Vaccinated donors were more likely than unvaccinated donors to neutralize Omicron when assessed by a PRNT50 assay. IMPORTANCE SARS-CoV-2 Omicron emergence occurred in Canada during the period from November 2021 to January 2022. This study assessed the ability of donor plasma collected earlier (January to March 2021) to generate any neutralizing capacity against Omicron BA.1 SARS-CoV-2. Vaccinated individuals, regardless of infection status, were more likely to neutralize Omicron BA.1 than unvaccinated individuals. This study then used a semiquantitative binding antibody assay to screen a larger number of specimens (4,453) for individual specimens that might have high-titer neutralizing capacity against Omicron BA.1. None of the 4,453 specimens tested by the semiquantitative SARS-CoV-2 assay had a binding capacity suggestive of a high-titer neutralizing capacity against Omicron BA.1. These data do not imply that Canadians lacked immunity to Omicron BA.1 during the study period. Immunity to SARS-CoV-2 is complex, and there is still no wide consensus on correlation of protection to SARS-CoV-2.

Patient and ward related risk factors in a multi-ward nosocomial outbreak of COVID-19: Outbreak investigation and matched case-control study.

BACKGROUND: Risk factors for nosocomial COVID-19 outbreaks continue to evolve. The aim of this study was to investigate a multi-ward nosocomial outbreak of COVID-19 between 1st September and 15th November 2020, occurring in a setting without vaccination for any healthcare workers or patients. METHODS: Outbreak report and retrospective, matched case-control study using incidence density sampling in three cardiac wards in an 1100-bed tertiary teaching hospital in Calgary, Alberta, Canada. Patients were confirmed/probable COVID-19 cases and contemporaneous control patients without COVID-19. COVID-19 outbreak definitions were based on Public Health guidelines. Clinical and environmental specimens were tested by RT-PCR and as applicable quantitative viral cultures and whole genome sequencing were conducted. Controls were inpatients on the cardiac wards during the study period confirmed to be without COVID-19, matched to outbreak cases by time of symptom onset dates, age within ± 15 years and were admitted in hospital for at least 2 days. Demographics, Braden Score, baseline medications, laboratory measures, co-morbidities, and hospitalization characteristics were collected on cases and controls. Univariate and multivariate conditional logistical regression was used to identify independent risk factors for nosocomial COVID-19. RESULTS: The outbreak involved 42 healthcare workers and 39 patients. The strongest independent risk factor for nosocomial COVID-19 (IRR 3.21, 95% CI 1.47-7.02) was exposure in a multi-bedded room. Of 45 strains successfully sequenced, 44 (97.8%) were B.1.128 and differed from the most common circulating community lineages. SARS-CoV-2 positive cultures were detected in 56.7% (34/60) of clinical and environmental specimens. The multidisciplinary outbreak team observed eleven contributing events to transmission during the outbreak. CONCLUSIONS: Transmission routes of SARS-CoV-2 in hospital outbreaks are complex; however multi-bedded rooms play a significant role in the transmission of SARS-CoV-2.

Utilization of the Abbott SARS-CoV-2 IgG II Quant Assay To Identify High-Titer Anti-SARS-CoV-2 Neutralizing Plasma against Wild-Type and Variant SARS-CoV-2 Viruses.

There is evidence that COVID-19 convalescent plasma may improve outcomes of patients with impaired immune systems; however, more clinical trials are required. Although we have previously used a 50% plaque reduction/neutralization titer (PRNT50) assay to qualify convalescent plasma for clinical trials and virus-like particle (VLP) assays to validate PRNT50 methodologies, these approaches are time-consuming and expensive. Here, we characterized the ability of the Abbott severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG II Quant assay to identify high- and low-titer plasma for wild-type and variant (Alpha, Beta, Gamma, and Delta) SARS-CoV-2 characterized by both VLP assays and PRNT50. Plasma specimens previously tested in wild-type, Alpha, Beta, Gamma, and Delta VLP neutralization assays were selected based on availability. Selected specimens were evaluated by the Abbott SARS-CoV-2 IgG II Quant assay [Abbott anti-Spike (S); Abbott, Chicago, IL], and values in units per milliliter were converted to binding antibody units (BAU) per milliliter. Sixty-three specimens were available for analysis. Abbott SARS-CoV-2 IgG II Quant assay values in BAU per milliliter were significantly different between high- and low-titer specimens for wild-type (Mann-Whitney U = 42, P < 0.0001), Alpha (Mann-Whitney U = 38, P < 0.0001), Beta (Mann-Whitney U = 29, P < 0.0001), Gamma (Mann-Whitney U = 0, P < 0.0001), and Delta (Mann-Whitney U = 42, P < 0.0001). A conservative approach using the highest 95% confidence interval (CI) values from wild-type and variant of concern (VOC) SARS-CoV-2 experiments would identify a potential Abbott SARS-CoV-2 IgG II Quant assay cutoff of ≥7.1 × 103 BAU/mL. IMPORTANCE The United States Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the use of COVID-19 convalescent plasma (CCP) to treat hospitalized patients with COVID-19 in August 2020. However, by 4 February 2021, the FDA had revised the convalescent plasma EUA. This revision limited the authorization for high-titer COVID-19 convalescent plasma and restricted patient groups to hospitalized patients with COVID-19 early in their disease course or hospitalized patients with impaired humoral immunity. Traditionally our group utilized 50% plaque reduction/neutralization titer (PRNT50) assays to qualify CCP in Canada. Since that time, the Abbott SARS-CoV-2 IgG II Quant assay (Abbott, Chicago IL) was developed for the qualitative and quantitative determination of IgG against the SARS-CoV-2. Here, we characterized the ability of the Abbott SARS-CoV-2 IgG II Quant assay to identify high- and low-titer plasma for wild-type and variant (Alpha, Beta, Gamma, and Delta) SARS-CoV-2.

The Role of Subgenomic RNA in Discordant Results From Reverse Transcription-Polymerase Chain Reaction Tests for COVID-19.

CONTEXT.­: Reverse transcription-polymerase chain reaction (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when 1 RT-PCR target is positive for SARS-CoV-2 and 1 RT-PCR target is negative for SARS-CoV-2 within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. OBJECTIVE.­: To understand the role of subgenomic RNA transcripts in discordant results from RT-PCR tests for COVID-19. DESIGN.­: A panel of 6 droplet digital PCR assays was designed to quantify the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity. Eleven clinical nasopharyngeal swabs were also tested with this panel. RESULTS.­: In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. The same trends in the relative abundance of copies across different targets observed in infected cells were observed in clinical samples, although trends were more pronounced in infected cells. CONCLUSIONS.­: This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by these data.

Detection and quantification of infectious severe acute respiratory coronavirus-2 in diverse clinical and environmental samples.

To explore the potential modes of Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) transmission, we collected 535 diverse clinical and environmental samples from 75 infected hospitalized and community patients. Infectious SARS-CoV-2 with quantitative burdens varying from 5 plaque-forming units/mL (PFU/mL) up to 1.0 × 106 PFU/mL was detected in 151/459 (33%) of the specimens assayed and up to 1.3 × 106 PFU/mL on fomites with confirmation by plaque morphology, PCR, immunohistochemistry, and/or sequencing. Infectious virus in clinical and associated environmental samples correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values ≤ 25 significantly predictive of yielding plaques in culture. SARS-CoV-2 isolated from patient respiratory tract samples caused illness in a hamster model with a minimum infectious dose of ≤ 14 PFU. Together, our findings offer compelling evidence that large respiratory droplet and contact (direct and indirect i.e., fomites) are important modes of SARS-CoV-2 transmission.

Viral load of SARS-CoV-2 in droplets and bioaerosols directly captured during breathing, speaking and coughing.

Determining the viral load and infectivity of SARS-CoV-2 in macroscopic respiratory droplets, bioaerosols, and other bodily fluids and secretions is important for identifying transmission modes, assessing risks and informing public health guidelines. Here we show that viral load of SARS-CoV-2 Ribonucleic Acid (RNA) in participants' naso-pharyngeal (NP) swabs positively correlated with RNA viral load they emitted in both droplets >10 [Formula: see text] and bioaerosols <10 [Formula: see text] directly captured during the combined expiratory activities of breathing, speaking and coughing using a standardized protocol, although the NP swabs had [Formula: see text] 10[Formula: see text] more RNA on average. By identifying highly-infectious individuals (maximum of 18,000 PFU/mL in NP), we retrieved higher numbers of SARS-CoV-2 RNA gene copies in bioaerosol samples (maximum of 4.8[Formula: see text] gene copies/mL and minimum cycle threshold of 26.2) relative to other studies. However, all attempts to identify infectious virus in size-segregated droplets and bioaerosols were negative by plaque assay (0 of 58). This outcome is partly attributed to the insufficient amount of viral material in each sample (as indicated by SARS-CoV-2 gene copies) or may indicate no infectious virus was present in such samples, although other possible factors are identified.

Prolonged SARS-CoV-2 infection following rituximab treatment: clinical course and response to therapeutic interventions correlated with quantitative viral cultures and cycle threshold values.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is completed through reverse transcriptase-PCR (RT-PCR) from either oropharyngeal or nasopharyngeal swabs, critically important for diagnostics but also from an infection control lens. Recent studies have suggested that COVID-19 patients can demonstrate prolonged viral shedding with immunosuppression as a key risk factor. CASE PRESENTATION: We present a case of an immunocompromised patient with SARS-CoV-2 infection demonstrating prolonged infectious viral shedding for 189 days with virus cultivability and clinical relapse with an identical strain based on whole genome sequencing, requiring a multi-modal therapeutic approach. We correlated clinical parameters, PCR cycle thresholds and viral culture until eventual resolution. CONCLUSIONS: We successfully demonstrate resolution of viral shedding, administration of COVID-19 vaccination and maintenance of viral clearance. This case highlights implications in the immunosuppressed patient towards infection prevention and control that should consider those with prolonged viral shedding and may require ancillary testing to fully elucidate viral activity. Furthermore, this case raises several stimulating questions around complex COVID-19 patients around the role of steroids, effect of antiviral therapies in absence of B-cells, role for vaccination and the requirement of a multi-modal approach to eventually have successful clearance of the virus.

Retention of hemostatic and immunological properties of frozen plasma and COVID-19 convalescent apheresis fresh-frozen plasma produced and freeze-dried in Canada.

BACKGROUND: Randomized clinical trial data show that early plasma transfusion may save lives among trauma patients. Supplying plasma in remote environments is logistically challenging. Freeze-dried plasma (FDP) offers a possible solution. STUDY DESIGN AND METHODS: A Terumo BCT plasma freeze-drying system was evaluated. We compared pooled frozen plasma (FP) units with derived Terumo BCT FDP (TFDP) units and pooled COVID-19 convalescent apheresis fresh-frozen plasma (CC-AFFP) with derived CC-TFDP units. Parameters measured were: coagulation factors (F) II; V; VII; VIII; IX; XI; XIII; fibrinogen; Proteins C (PC) and S (PS); antithrombin (AT); α2 -antiplasmin (α2 AP); ADAMTS13; von Willebrand Factor (vWF); thrombin-antithrombin (TAT); D-dimer; activated complement factors 3 (C3a) and 5 (C5a); pH; osmolality; prothrombin time (PT); and activated partial thromboplastin time (aPTT). Antibodies to SARS-CoV-2 in CC-AFFP and CC-TFDP units were compared by plaque reduction assays and viral protein immunoassays. RESULTS: Most parameters were unchanged in TFDP versus FP or differed ≤15%. Mean aPTT, PT, C3a, and pH were elevated 5.9%, 6.9%, 64%, and 0.28 units, respectively, versus FP. CC-TFDP showed no loss of SARS-CoV-2 neutralization titer versus CC-AFFP and no mean signal loss in most pools by viral protein immunoassays. CONCLUSION: Changes in protein activities or clotting times arising from freeze-drying were <15%. Although C3a levels in TFDP were elevated, they were less than literature values for transfusable plasma. SARS-CoV-2-neutralizing antibody titers and viral protein binding levels were largely unaffected by freeze-drying. In vitro characteristics of TFDP or CC-TFDP were comparable to their originating plasma, making future clinical studies appropriate.

Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Resistance of SARS-CoV-2 beta and gamma variants to plasma collected from Canadian blood donors during the spring of 2020.

BACKGROUND: This pilot study assesses the ability of plasma collected from Canadian blood donors in the first wave of the SARS-CoV-2 pandemic to neutralize later SARS-CoV-2 variants of concern (VOCs). STUDY DESIGN AND METHODS: A repeated cross-sectional design was used, and a random cross-sectional sample of all available Canadian Blood Services retention samples (n = 1500/month) was drawn monthly for April and May of 2020. Qualitative IgG analysis was performed on aliquots of specimens using anti-spike, anti-receptor binding domain, and anti-nucleocapsid protein enzyme-linked immunosorbent assays as well as the Abbott Architect SARS CoV-2 IgG assay (Abbott Laboratories) against the anti-nucleocapsid protein. Selected plasma specimens were then assessed for neutralization against VOCs using pseudotyped lentivirus inhibition assays as well as plaque reduction neutralization test 50% (PRNT50 ). RESULTS: Six specimens with a high neutralizing titer against wild-type SARS-CoV-2 and three specimens with a low neutralizing titer against wild-type SARS-CoV-2 were chosen for further analysis against VOCs. Four of six high neutralizing titer specimens had a reduced neutralizing capacity against beta VOCs by both neutralization methods. Three of six high neutralizing titer specimens had reduced neutralization capacity against gamma VOCs. CONCLUSIONS: This preliminary data can be used as a justification for limiting the use of first wave plasma products in upcoming clinical trials but cannot be used to speculate on general trends in the immunity of Canadian blood donors to SARS-CoV-2.

Addressing personal protective equipment (PPE) decontamination: Methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus. DESIGN: The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance. METHODS: MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. RESULTS: Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3. CONCLUSIONS: MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.