Detection of SARS-CoV-2 variants by Abbott molecular, antigen, and serological tests (preprint)

Background Viral diversity presents an ongoing challenge for diagnostic tests, which need to accurately detect all circulating variants. The Abbott Global Surveillance program monitors severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and their impact on diagnostic test performance. Objectives To evaluate the capacity of Abbott molecular, antigen, and serologic assays to detect the SARS-CoV-2 B.1.1.7, B.1.351 and the P.1 variants. Study design Virus variant culture stock dilutions (B.1.1.7, BEI NR-54011; B.1.351, BEI NR-54008 and 54009; P.1, BEI NR-54982) and clinical samples from patients with confirmed B.1.1.7 variant infection were run on the Abbott ID NOW COVID-19, m 2000 RealTi m e SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex molecular assays; the BinaxNOW COVID-19 Ag Card and Panbio COVID-19 Ag Rapid Test Device; and the ARCHITECT/Alinity i SARS-CoV-2 IgG and AdviseDx IgM assays, Panbio COVID-19 IgG assay, and ARCHITECT/Alinity i AdviseDx SARS-CoV-2 IgG II assay. Results Cultured virus stocks and B.1.1.7 clinical samples were detected with molecular, antigen, and serologic assays in the expected ranges, confirming in silico predictions. The ratio between genome equivalents (GE) and calculated median tissue culture infectious dose (TCID 50 ) were 31-to 83-fold higher for B.1.1.7 cultures compared to B.1.351 and P.1 cultures, demonstrating that GE are more consistent units between cultures than TCID 50 . Conclusions Abbott molecular and antigen assays effectively detect B.1.1.7, B.1.351, and P.1 variant infections and Abbott serologic assays detect B.1.1.7 antibodies in patient sera. Future studies with SARS-CoV-2 virus cultures should use quantitative viral load values to compare detection of variants. Highlights Abbott SARS-CoV-2 molecular and antigen assays detect B.1.1.7, B.1.351, and P.1 variants Abbott SARS-CoV-2 antibody assays detect B.1.1.7 antibodies in recovered patient sera Quantitation of viral load in genome equivalents allows comparison of assay performance

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

Background: Rapid 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. Methods: A 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. Findings: Most 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 3x106/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. Interpretation: In 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.

Clinical Outcomes of Patients With and Without HIV Hospitalised with COVID-19 in England During the Early Stages of the Pandemic: A Matched Retrospective Multicentre Analysis (RECEDE-C19 Study) (preprint)

Background: It is unclear from epidemiological data for COVID-19 infections, whether people living with HIV (PLWH) have a different outcomes compared to the general population. We conducted a multi-centre, retrospective matched cohort study of SARS-CoV-2 PCR-positive hospital inpatients analysed by HIV-status.Methods: HIV-negative patients were matched to PLWH admitted to hospital before 31 st May 2020, with a 3:1 ratio by: hospital site, SARS-CoV-2 test date +/- 7 days, age +/- 5 years, gender, and index of multiple deprivation decile (IMDD) +/- 1. The primary objective was clinical improvement (≥2-point improvement on a 7-point ordinal scale) or hospital discharge by day 28, whichever was earlier.Results: 68 PLWH and 181 HIV-negative comparators were included. After adjustment for ethnicity, frailty, baseline hypoxia, duration of symptoms prior to baseline, body mass index categories, and comorbidities (hypertension, chronic cardiac disease, chronic lung disease, active malignancy, diabetes, and chronic renal disease), the effect size of HIV status was not associated with time to clinical improvement or discharge from hospital (aHR 0.70, 95%CI 0.43, 1.17; p=0.18), despite unadjusted hazards of PLWH achieving the primary outcome being 43% lower (p=0.005). Baseline frailty (aHR=0.79; 95%CI 0.65, 0.95; p=0.011), malignancy (aHR=0.37; 95%CI 0.17, 0.82; p=0.014) remained associated with poorer outcomes. PLWH were more likely of black and minority ethnicities (75.0% vs 48.6%, p=0.0002), higher median clinical frailty score (3 IQR 2-5 vs 2 IQR 1-4, p=0.0069), higher proportion of active malignancy (14.4% vs 9.9%, p=0.29). Median body mass index (BMI) was lower amongst PLWH (27.7 IQR 23.9-32.3 vs 29.4 IQR 24.7-34.3, p=0.19). Median CD4 count of PLWH was 352cells/µL (IQR 235-619) and 95.7% had suppressed viral loads <200copies/mL, 63/68 (92.3%) were taking antiretroviral therapy.Conclusions: Differences in clinical outcomes of COVID-19 hospitalisations in PLWH may be due to other important factors including increased frailty and comorbidities such as malignancies, rather than HIV-status alone.Funding Statement: This study has not received any funding sources.Declaration of Interests: MJL has received grants and honoraria from Gilead Sciences and Viiv Healthcare not related to this work. SF has received research grants to her institution from NIH, MRC, BMGF. JT has received support for virtual conference registration from ViiV Healthcare and research grants from the Medical Research Council and the British HIV Association not related to this work. CvH has received educational grants, conference support and advisory board fees from ViiV Healthcare, Gilead Sciences, MSC not related to this work. MP reports grants and personal fees from Gilead Sciences and personal fees from QIAGEN, outside the submitted work. MP is supported by a NIHR Development and Skills Enhancement Award (NIHR301192) and in receipt of funding from UKRI / MRC (MR/V027549/1). He acknowledges the support from UKRI, the NIHR Leicester BRC and NIHR ARC East Midlands. No other competing interests, financial relationships with any organisations that might have an interest in the submitted work, or other relationships or activities that could appear to have influenced the submitted work have been reported by other authors.Ethics Approval Statement: Ethical approval was granted by the UK Health Research Authority (REC reference 20/HRA/2278).

Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection (preprint)

Antibody (Ab) responses to SARS-CoV-2 can be detected in most infected individuals 10-15 days following the onset of COVID-19 symptoms. However, due to the recent emergence of this virus in the human population it is not yet known how long these Ab responses will be maintained or whether they will provide protection from re-infection. Using sequential serum samples collected up to 94 days post onset of symptoms (POS) from 65 RT-qPCR confirmed SARS-CoV-2-infected individuals, we show seroconversion in >95% of cases and neutralizing antibody (nAb) responses when sampled beyond 8 days POS. We demonstrate that the magnitude of the nAb response is dependent upon the disease severity, but this does not affect the kinetics of the nAb response. Declining nAb titres were observed during the follow up period. Whilst some individuals with high peak ID50 (>10,000) maintained titres >1,000 at >60 days POS, some with lower peak ID50 had titres approaching baseline within the follow up period. A similar decline in nAb titres was also observed in a cohort of seropositive healthcare workers from Guys and St Thomas Hospitals. We suggest that this transient nAb response is a feature shared by both a SARS-CoV-2 infection that causes low disease severity and the circulating seasonal coronaviruses that are associated with common colds. This study has important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection.

Clinical utility of targeted SARS-CoV-2 serology testing to aid the diagnosis and management of suspected missed, late or post-COVID-19 infection syndromes: results from a pilot service (preprint)

Objectives: Determine indications and clinical utility of SARS-CoV-2 serology testing in adults and children. Design: Prospective evaluation of initial three weeks of a daily Monday to Friday pilot SARS-CoV-2 serology service for patients. Setting: Early post 'first-wave' SARS-CoV-2 transmission period at single centre London teaching hospital that provides care to the local community, as well as regional and national referral pathways for specialist services. Participants: 110 (72 adults, 38 children, age range 0-83 years, 52.7% female (n=58)). Interventions: Patient serum from vetted referrals tested on CE marked and internally validated lateral flow immunoassay (LFIA) (SureScreen Diagnostics) detecting antibodies to SARS-CoV-2 spike proteins, with result and clinical interpretation provided to the direct care team. Main outcome measures: Performance characteristics, source and nature of referrals, feasibility and clinical utility of the service, particularly the benefit for clinical decision-making. Results: The LFIA was deemed suitable for clinical advice and decision making following evaluation with 310 serum samples from SARS-CoV-2 PCR positive patients and 300 pre-pandemic samples, giving a sensitivity and specificity of 96.1% and 99.3% respectively. For the pilot, 115 referrals were received leading to 113 tests performed on 108 participants (sample not available for two participants); paediatrics (n=35), medicine (n=69), surgery (n=2) and general practice (n=2). 43.4% participants (n=49) had detectable antibodies to SARS-CoV-2. There were three main indications for serology; new acute presentations potentially triggered by recent COVID-19 infection e.g. PIMS-TS (n=26) and pulmonary embolism (n=5), potential missed diagnoses in context of a recent compatible illness (n=40), and making infection control and immunosuppression treatment decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n=6). Conclusions: This study shows acceptable performance characteristics, feasibility and clinical utility of a SARS-CoV-2 serology service using a rapid, inexpensive and portable assay for adults and children presenting with a range of clinical indications. Results correlated closely with a confirmatory in-house ELISA. The study showed the benefit of introducing a serology service where there is a reasonable pre-test probability, and the result can be linked with clinical advice or intervention. Experience thus far is that the volume of requests from hospital referral routes are manageable within existing clinical and laboratory services; however, the demand from community referrals has not yet been assessed. Given recent evidence for a rapid decline in antibodies, particularly following mild infection, there is likely a limited window of opportunity to realise the benefit of serology testing for individuals infected during the 'first-wave' before they potentially fall below a measurable threshold. Rapidly expanding availability of serology services for NHS patients will also help understand the long-term implications of serostatus and prior infection in different patient groups, particularly before emergence of any 'second-wave' outbreak or introduction of a vaccination programme.

Comparative assessment of multiple COVID-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings (preprint)

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential as diagnostic tools. A highly specific in-house ELISA was developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays - a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs) - on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.

Detecting SARS-CoV-2 at point of care: Preliminary data comparing Loop-mediated isothermal amplification (LAMP) to PCR (preprint)

Background: The need for a fast and reliable test for COVID-19 is paramount in managing the current pandemic. A cost effective and efficient diagnostic tool as near to the point of care (PoC) as possible would be a game changer in current testing. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 minutes, alongside standard methods in a real-life clinical setting. Methods: This service improvement project piloted a research RT-LAMP method on nasal and pharyngeal swabs on 21 residents in an NHS Category 1 care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We calculated the sensitivity, specificity, positive and negative predictive values of a single RT-LAMP swab compared to RT-PCR, as per STARD guidelines. We also recorded vital signs of patients to correlate clinical and laboratory information. Findings: The novel method accurately detected 8/10 PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a 'gold standard', the sensitivity and specificity of the novel test were 80% and 73% respectively. Positive predictive value (PPV) was 73% and negative predictive value (NPV) was 83%. We also observed hypothermia to be a significant early clinical sign in a number of COVID-19 patients in this setting. Interpretation: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast, easy to use and to work equivalently to RT-PCR methods. Definitive studies to evaluate this method in larger cohorts are underway. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the point of care. This method could be deployed in mobile testing units in the community, care homes and hospitals to detect disease early and prevent spread.