Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow assay for antibody prevalence studies following vaccination: a diagnostic accuracy study.

Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay Results: The LFIA had an estimated sensitivity of 92.0% (114/124; 95% confidence interval [CI] 85.7% to 96.1%) and specificity of 93.6% (58/62; 95% CI 84.3% to 98.2%) using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml) Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow assay for antibody prevalence studies following vaccination: a diagnostic accuracy study [version 2;peer review: 2 approved]

Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited;the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay Results: The LFIA had an estimated sensitivity of 92.0% (114/124;95% confidence interval [CI] 85.7% to 96.1%) and specificity of 93.6% (58/62;95% CI 84.3% to 98.2%) using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml) Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing.

Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study.

BACKGROUND: Access to rapid diagnosis is key to the control and management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Laboratory RT-PCR testing is the current standard of care but usually requires a centralised laboratory and significant infrastructure. We describe our diagnostic accuracy assessment of a novel, rapid point-of-care real time RT-PCR CovidNudge test, which requires no laboratory handling or sample pre-processing. METHODS: Between April and May, 2020, we obtained two nasopharyngeal swab samples from individuals in three hospitals in London and Oxford (UK). Samples were collected from three groups: self-referred health-care workers with suspected COVID-19; patients attending emergency departments with suspected COVID-19; and hospital inpatient admissions with or without suspected COVID-19. For the CovidNudge test, nasopharyngeal swabs were inserted directly into a cartridge which contains all reagents and components required for RT-PCR reactions, including multiple technical replicates of seven SARS-CoV-2 gene targets (rdrp1, rdrp2, e-gene, n-gene, n1, n2 and n3) and human ribonuclease P (RNaseP) as sample adequacy control. Swab samples were tested in parallel using the CovidNudge platform, and with standard laboratory RT-PCR using swabs in viral transport medium for processing in a central laboratory. The primary analysis was to compare the sensitivity and specificity of the point-of-care CovidNudge test with laboratory-based testing. FINDINGS: We obtained 386 paired samples: 280 (73%) from self-referred health-care workers, 15 (4%) from patients in the emergency department, and 91 (23%) hospital inpatient admissions. Of the 386 paired samples, 67 tested positive on the CovidNudge point-of-care platform and 71 with standard laboratory RT-PCR. The overall sensitivity of the point-of-care test compared with laboratory-based testing was 94% (95% CI 86-98) with an overall specificity of 100% (99-100). The sensitivity of the test varied by group (self-referred healthcare workers 94% [95% CI 85-98]; patients in the emergency department 100% [48-100]; and hospital inpatient admissions 100% [29-100]). Specificity was consistent between groups (self-referred health-care workers 100% [95% CI 98-100]; patients in the emergency department 100% [69-100]; and hospital inpatient admissions 100% [96-100]). Point of care testing performance was similar during a period of high background prevalence of laboratory positive tests (25% [95% 20-31] in April, 2020) and low prevalence (3% [95% 1-9] in inpatient screening). Amplification of viral nucleocapsid (n1, n2, and n3) and envelope protein gene (e-gene) were most sensitive for detection of spiked SARS-CoV-2 RNA. INTERPRETATION: The CovidNudge platform was a sensitive, specific, and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. The device, which has been implemented in UK hospitals since May, 2020, could enable rapid decisions for clinical care and testing programmes. FUNDING: National Institute of Health Research (NIHR) Imperial Biomedical Research Centre, NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University in partnership with Public Health England, NIHR Biomedical Research Centre Oxford, and DnaNudge.

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

SARS-CoV-2 lateral flow assays for possible use in national covid-19 seroprevalence surveys (React 2): diagnostic accuracy study.

OBJECTIVE: To evaluate the performance of new lateral flow immunoassays (LFIAs) suitable for use in a national coronavirus disease 2019 (covid-19) seroprevalence programme (real time assessment of community transmission 2-React 2). DESIGN: Diagnostic accuracy study. SETTING: Laboratory analyses were performed in the United Kingdom at Imperial College, London and university facilities in London. Research clinics for finger prick sampling were run in two affiliated NHS trusts. PARTICIPANTS: Sensitivity analyses were performed on sera stored from 320 previous participants in the React 2 programme with confirmed previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Specificity analyses were performed on 1000 prepandemic serum samples. 100 new participants with confirmed previous SARS-CoV-2 infection attended study clinics for finger prick testing. INTERVENTIONS: Laboratory sensitivity and specificity analyses were performed for seven LFIAs on a minimum of 200 serum samples from participants with confirmed SARS-CoV-2 infection and 500 prepandemic serum samples, respectively. Three LFIAs were found to have a laboratory sensitivity superior to the finger prick sensitivity of the LFIA currently used in React 2 seroprevalence studies (84%). These LFIAs were then further evaluated through finger prick testing on participants with confirmed previous SARS-CoV-2 infection: two LFIAs (Surescreen, Panbio) were evaluated in clinics in June-July 2020 and the third LFIA (AbC-19) in September 2020. A spike protein enzyme linked immunoassay and hybrid double antigen binding assay were used as laboratory reference standards. MAIN OUTCOME MEASURES: The accuracy of LFIAs in detecting immunoglobulin G (IgG) antibodies to SARS-CoV-2 compared with two reference standards. RESULTS: The sensitivity and specificity of seven new LFIAs that were analysed using sera varied from 69% to 100%, and from 98.6% to 100%, respectively (compared with the two reference standards). Sensitivity on finger prick testing was 77% (95% confidence interval 61.4% to 88.2%) for Panbio, 86% (72.7% to 94.8%) for Surescreen, and 69% (53.8% to 81.3%) for AbC-19 compared with the reference standards. Sensitivity for sera from matched clinical samples performed on AbC-19 was significantly higher with serum than finger prick at 92% (80.0% to 97.7%, P=0.01). Antibody titres varied considerably among cohorts. The numbers of positive samples identified by finger prick in the lowest antibody titre quarter varied among LFIAs. CONCLUSIONS: One new LFIA was identified with clinical performance suitable for potential inclusion in seroprevalence studies. However, none of the LFIAs tested had clearly superior performance to the LFIA currently used in React 2 seroprevalence surveys, and none showed sufficient sensitivity and specificity to be considered for routine clinical use.

Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey.

BACKGROUND: Accurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required. METHODS: Sensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by reverse transcription PCR and were ≥21 days from symptom onset. In phase I, we evaluated five LFIAs in clinic (with finger prick) and laboratory (with blood and sera) in comparison to (1) PCR-confirmed infection and (2) presence of SARS-CoV-2 antibodies on two 'in-house' ELISAs. Specificity analysis was performed on 500 prepandemic sera. In phase II, six additional LFIAs were assessed with serum. FINDINGS: 95% (95% CI 92.2% to 97.3%) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8 out of 11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21% to 92% versus PCR-confirmed cases and from 22% to 96% versus composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2%-99.8%). INTERPRETATION: LFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI 97.1% to 99.4%)), moderate sensitivity (84.4% with finger prick (95% CI 70.5% to 93.5%)) and moderate concordance, suitable for seroprevalence surveys.