Clinical evaluation of the SD Biosensor SARS-CoV-2 saliva antigen rapid test with symptomatic and asymptomatic, non-hospitalized patients

Background Performance of the SD Biosensor saliva antigen rapid test was evaluated at a large designated testing site in non-hospitalized patients, with or without symptoms. Method All eligible people over 18 years of age presenting for a booked appointment at the designated SARS-CoV-2 testing site were approached for inclusion and enrolled following verbal informed consent. One nasopharyngeal swab was taken to carry out the default antigen rapid test from which the results were reported back to the patient and one saliva sample was self-taken according to verbal instruction on site. This was used for the saliva antigen rapid test, the RT-PCR and for virus culture. Sensitivity of the saliva antigen rapid test was analyzed in two ways: i, compared to saliva RT-PCR; and ii, compared to virus culture of the saliva samples. Study participants were also asked to fill in a short questionnaire stating age, sex, date of symptom onset. Recommended time of ≥30mins since last meal, drink or cigarette if applicable was also recorded. The study was carried out in February-March 2021 for 4 weeks. Results We could include 789 people with complete records and results. Compared to saliva RT-PCR, overall sensitivity and specificity of the saliva antigen rapid test was 66.1% and 99.6% which increased to 88.6% with Ct ≤30 cutoff. Analysis by days post onset did not result in higher sensitivities because the large majority of people were in the very early phase of disease ie <3 days post onset. When breaking down the data for symptomatic and asymptomatic individuals, sensitivity ranged from 69.2% to 50% respectively, however the total number of RT-PCR positive asymptomatic participants was very low (n = 5). Importantly, almost all culture positive samples were detected by the rapid test. Conclusion Overall, the potential benefits of saliva antigen rapid test, could outweigh the lower sensitivity compared to nasopharyngeal antigen rapid test in a comprehensive testing strategy, especially for home/self-testing and in vulnerable populations like elderly, disabled or children where in intrusive testing is either not possible or causes unnecessary stress.

Virological characteristics of SARS-CoV-2 vaccine breakthrough infections in health care workers

SARS-CoV-2 vaccines are highly effective at preventing COVID-19-related morbidity and mortality. As no vaccine is 100% effective, breakthrough infections are expected to occur. We analyzed the virological characteristics of 161 vaccine breakthrough infections in a population of 24,706 vaccinated healthcare workers (HCWs), using RT-PCR and virus culture. The delta variant (B.1.617.2) was identified in the majority of cases. Despite similar Ct-values, we demonstrate lower probability of infectious virus detection in respiratory samples of vaccinated HCWs with breakthrough infections compared to unvaccinated HCWs with primary SARS-CoV-2 infections. Nevertheless, infectious virus was found in 68.6% of breakthrough infections and Ct-values decreased throughout the first 3 days of illness. We conclude that rare vaccine breakthrough infections occur, but infectious virus shedding is reduced in these cases.

Primary Immune Response Provides Protective Efficacy against SARS-CoV-2 Reinfection

While there is absolutely no evidence to ensure recovered patients are either likely or unlikely to get reinfected. But studies in non-human primates indicate that reinfection of recovered patients is highly unlikely. It is also clear that primary immune responses or induced immunity to severe acute respiratory syndrome coronavirus 2 remain in circulation for several months and at least temporarily confer immunity to protect from reinfection. In addition, negative virus culture analysis of re-positive suggests that positive reverse transcriptase-polymerase chain reactions in recovered patients are more likely to be false-positive, or detection of genetic remnants of virus discharged from lesions of lungs or better sampling at the time of repeat analysis. However, emerging severe acute respiratory syndrome coronavirus 2 variants are likely to be causing the infections observed in some of the recovered patients.

Evaluation of a rapid antigen test to detect SARS-CoV-2 infection and identify potentially infectious individuals

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising candidates for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is yet to be fully determined. In this study, RT-qPCR and virus culture of RT-qPCR positive samples were used to evaluate and compare the performance of the Standard Q COVID-19 Ag Test in detecting SARS-CoV-2 infected and possibly infectious individuals. To this end, two combined oro- and nasopharyngeal swabs were collected at a routine SARS-CoV-2 diagnostic center. A total of 2,028 samples were tested and 118 virus cultures inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag Test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86% and 99.89%. For adjusted Ct values <20 (n=14), <25 (n=57), and <30 (n=88) the RADT reached sensitivities of 100%, 98.25%, and 88.64%, respectively. All 29 culture positive samples were detected by RADT. While overall sensitivity was low, Standard Q COVID-19 RADT reliably detected patients with high RNA loads. Additionally, negative RADT results fully corresponded with the lack of viral cultivability in Vero E6 cells. These results indicate that RADT can be a valuable tool for the detection of individuals that are likely to transmit SARS-CoV-2. RADT testing could therefore guide public health testing strategies to combat the COVID-19 pandemic.

Clinical evaluation of the SD Biosensor saliva antigen rapid test with symptomatic and asymptomatic, non-hospitalized patients

Performance of saliva antigen rapid test was evaluated in non-hospitalized patients, with or without symptoms. Overall sensitivity was 66.1% compared to RT-PCR in saliva. Using cycle threshold <30 cutoff or virus culture as reference, sensitivity increased to 88.6% or 96.7% respectively. Specificity was 99.6%.

Delayed rise of oral fluid antibodies, elevated BMI, and absence of early fever correlate with longer time to SARS-CoV-2 RNA clearance in a longitudinally sampled cohort of COVID-19 outpatients

Background Sustained molecular detection of SARS-CoV-2 RNA in the upper respiratory tract (URT) in mild to moderate COVID-19 is common. We sought to identify host and immune determinants of prolonged SARS-CoV-2 RNA detection. Methods Ninety-five symptomatic outpatients self-collected mid-turbinate nasal, oropharyngeal (OP), and gingival crevicular fluid (oral fluid) samples at home and in a research clinic a median of 6 times over 1-3 months. Samples were tested for viral RNA, virus culture, and SARS-CoV-2 and other human coronavirus antibodies, and associations were estimated using Cox proportional hazards models. Results Viral RNA clearance, as measured by SARS-CoV-2 RT-PCR, in 507 URT samples occurred a median (IQR) 33.5 (17-63.5) days post-symptom onset. Sixteen nasal-OP samples collected 2-11 days post-symptom onset were virus culture positive out of 183 RT-PCR positive samples tested. All participants but one with positive virus culture were negative for concomitant oral fluid anti-SARS-CoV-2 antibodies. The mean time to first antibody detection in oral fluid was 8-13 days post-symptom onset. A longer time to first detection of oral fluid anti-SARS-CoV-2 S antibodies (aHR 0.96, 95% CI 0.92-0.99, p=0.020) and BMI ≥ 25kg/m2 (aHR 0.37, 95% CI 0.18-0.78, p=0.009) were independently associated with a longer time to SARS-CoV-2 viral RNA clearance. Fever as one of first three COVID-19 symptoms correlated with shorter time to viral RNA clearance (aHR 2.06, 95% CI 1.02-4.18, p=0.044). Conclusions We demonstrate that delayed rise of oral fluid SARS-CoV-2-specific antibodies, elevated BMI, and absence of early fever are independently associated with delayed URT viral RNA clearance.

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

Delayed rise of oral fluid antibodies, elevated BMI, and absence of early fever correlate with longer time to SARS-CoV-2 RNA clearance in an longitudinally sampled cohort of COVID-19 outpatients

ABSTRACTBackgroundSustained molecular detection of SARS-CoV-2 RNA in the upper respiratory tract (URT) in mild to moderate COVID-19 is common. We sought to identify host and immune determinants of prolonged SARS-CoV-2 RNA detection.MethodsNinety-five outpatients self-collected mid-turbinate nasal, oropharyngeal (OP), and gingival crevicular fluid (oral fluid) samples at home and in a research clinic a median of 6 times over 1-3 months. Samples were tested for viral RNA, virus culture, and SARS-CoV-2 and other human coronavirus antibodies, and associations were estimated using Cox proportional hazards models.ResultsViral RNA clearance, as measured by SARS-CoV-2 RT-PCR, in 507 URT samples occurred a median (IQR) 33.5 (17-63.5) days post-symptom onset. Sixteen nasal-OP samples collected 2-11 days post-symptom onset were virus culture positive out of 183 RT-PCR positive samples tested. All participants but one with positive virus culture were negative for concomitant oral fluid anti-SARS-CoV-2 antibodies. The mean time to first antibody detection in oral fluid was 8-13 days post-symptom onset. A longer time to first detection of oral fluid anti-SARS-CoV-2 S antibodies (aHR 0.96, 95% CI 0.92-0.99, p=0.020) and BMI ≥ 25kg/m2 (aHR 0.37, 95% CI 0.18-0.78, p=0.009) were independently associated with a longer time to SARS-CoV-2 viral RNA clearance. Fever as one of first three COVID-19 symptoms correlated with shorter time to viral RNA clearance (aHR 2.06, 95% CI 1.02-4.18, p=0.044).ConclusionsWe demonstrate that delayed rise of oral fluid SARS-CoV-2-specific antibodies, elevated BMI, and absence of early fever are independently associated with delayed URT viral RNA clearance.

Comparative performance of SARS CoV-2 lateral flow antigen tests demonstrates their utility for high sensitivity detection of infectious virus in clinical specimens

BackgroundRapid antigen lateral flow devices (LFDs) are set to become a cornerstone of SARS-CoV-2 mass community testing. However, their reduced sensitivity compared to PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is therefore essential for successful implementation. To address this, we evaluated six commercial LFDs on the same collection of clinical samples and assessed their correlation with infectious virus culture and cycle threshold (Ct) values.MethodsA head-to-head comparison of specificities and sensitivities was performed on six commercial rapid antigen tests using combined nasal/oropharyngeal swabs, and their limits of detection determined using viral plaque forming units (PFU). Three of the LFDs were selected for a further study, correlating antigen test result with RT-PCR Ct values and positive viral culture in Vero-E6 cells. This included sequential swabs and matched serum samples obtained from four infected individuals with varying disease severities. Detection of antibodies was performed using an IgG/IgM Rapid Test Cassette, and neutralising antibodies by infectious virus assay. Finally, the sensitivities of selected rapid antigen LFTs were assessed in swabs with confirmed B.1.1.7 variant, currently the dominant genotype in the UK.FindingsMost of the rapid antigen LFDs showed a high specificity (>98%), and accurately detected 50 PFU/test (equivalent N1 Ct of 23.7 or RNA copy number of 3×106/ml). Sensitivities of the LFDs performed on clinical samples ranged from 65 to 89%. These sensitivities increased in most tests to over 90% for samples with Cts lower than 25. Positive virus culture was achieved for 57 out of 141 samples, with 80% of the positive cultures from swabs with Cts lower than 23. Importantly, sensitivity of the LFDs increased to over 95% when compared with the detection of infectious virus alone, irrespective of Ct. Longitudinal studies of PCR-positive samples showed that most of the tests identified all infectious samples as positive, but differences in test sensitivities can lead to missed cases in the absence of repeated testing. Finally, test performance was not impacted when re-assessed against swabs positive for the dominant UK variant B.1.1.7.InterpretationIn this comprehensive comparison of antigen LFD and virus infectivity, we demonstrate a clear relationship between Ct values, quantitative culture of infectious virus and antigen LFD positivity in clinical samples. Our data support regular testing of target groups using LFDs to supplement the current PCR testing capacity, to rapidly identify infected individuals in situations where they would otherwise go undetected.FundingKing’s Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation.