More than the eye can see; shedding new light on SARS-CoV-2 Lateral Flow Device-based immunoassays (preprint)

Containing the global SARS-CoV-2 pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral Flow Device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or pre-symptomatic individuals rapidly. Whilst LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or pre-symptomatic carriers may wrongly be assumed to be non-infectious, posing a significant risk of further spread in the community. Here we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a bio-sample. We quantified significant immobilized antigen-antibody-label conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and qRT-PCR determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to [~]10,000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance, both to public health monitoring during the COVID-19 pandemic and to the rapid refinement of these tools for immediate and future applications.

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.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation (preprint)

There is a worldwide shortage of reagents to perform detection of SARS-2. Many clinical diagnostic laboratories rely on commercial platforms that provide integrated end-to-end solutions. While this provides established robust pipelines, there is a clear bottleneck in the supply of reagents given the current situation of extraordinary high demand. Some laboratories resort to implementing kit-free handling procedures, but many other small laboratories will not have the capacity to develop those and/or will perform manual handling of their samples. In order to provide multiple workflows for SARS-CoV-2 nucleic acid detection we compared several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), the recently developed RNAdvance Blood (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared different 1-step RT-qPCR Master Mix brands: TaqMan Fast Virus 1-Step Master Mix (ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna(R) Universal Probe One-Step RT-qPCR Kit (NEB). We used the Centre for Disease Control (CDC) recommended primers that detect two regions of the viral N gene as well as those that detect the RdRP gene region as per Public Health England (PHE) guidelines (Charite/WHO/PHE). Our data show that the RNA extraction methods provide similar results. Amongst the qPCR reagents tested, TaqMan Fast Virus 1-Step Master Mix and Luna(R) Universal Probe One-Step RT-qPCR Kit proved most sensitive. The N1 and N2 primer-probes provide a more reliable detection than the RdRP-SARSr primer-probe set, particularly in samples with low viral titres. Importantly, we have implemented a protocol using heat inactivation and demonstrate that it has minimal impact on the sensitivity of the qPCR in clinical samples - potentially making SARS-CoV-2 testing portable to settings that do not have CL-3 facilities.