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.

Application of respiratory metagenomics for COVID-19 patients on the intensive care unit to inform appropriate initial antimicrobial treatment and rapid detection of nosocomial transmission (preprint)

Background. Clinical metagenomics (CMg) is being evaluated for translation from a research tool into routine diagnostic service, but its potential to significantly improve management of acutely unwell patients has not been demonstrated. The SARS-CoV-2 pandemic provides impetus to determine that benefit given increased risk of secondary infection and nosocomial transmission by multi-drug resistant (MDR) pathogens linked with expansion of critical care capacity. Methods. Prospective evaluation of CMg using nanopore sequencing was performed on 43 respiratory samples over 14 weeks from a cohort of 274 intubated patients across seven COVID-19 intensive care units. Results. Bacteria or fungi were cultured from 200 (73%) patients, with a predominance of Klebsiella spp. (31%) and C. striatum (7%) amongst other common respiratory pathogens. An 8 hour CMg workflow was 93% sensitive and 81% specific for bacterial identification compared to culture, and reported presence or absence of {beta}-lactam resistance genes carried by Enterobacterales that would modify initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus (4 positive and 39 negative samples). Single nucleotide polymorphism (SNP)-typing using 24 hour sequence data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak potentially involving 14 patients across three ICUs. Conclusion. CMg testing for ICU patients provides same-day pathogen detection and antibiotic resistance prediction that significantly improves initial treatment of nosocomial pneumonia and rapidly detects unsuspected outbreaks of MDR-pathogens.

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.

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.