RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals (preprint)

Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal/oropharyngeal swab and saliva samples. Here we describe the validation of an improved simple sample preparation method for Direct SARS-CoV-2 RT-LAMP, removing the need for RNA extraction, using 559 swabs and 86,760 saliva samples from asymptomatic and symptomatic individuals across multiple healthcare settings. Using this improved method we report a diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva, with diagnostic specificity (DSp) 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR. Analysing samples with RT-qPCR ORF1ab CT values of <25 and <33 (high and medium-high viral loads, respectively), we found DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs, and 99.01% (94.61-99.97%) and 87.32% (80.71-92.31%) for saliva. We also describe RNA RT-LAMP (on extracted RNA) performed on 12,619 swabs and 12,521 saliva samples to provide updated performance data with DSe and DSp of 95.98% (92.74-98.06%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. We also report on daily samples collected from one individual from symptom onset where both Direct and RNA RT-LAMP detected SARS-CoV-2 in saliva collected on all six days where symptoms were recorded, with RNA RT-LAMP detecting SARS-CoV-2 for an additional further day. The findings from these studies demonstrate that RT-LAMP testing of swabs and saliva is potentially applicable to a variety of use-cases, including frequent, interval-based testing of saliva from asymptomatic individuals via Direct RT-LAMP that may be missed using symptomatic testing alone.

Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions (preprint)

IntroductionFaecal transplantation is an evidence based treatment for Clostridiodes difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for FMT, spiked samples and asymptomatic inpatients in an acute surgical unit. MethodsKilled SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10-1 to 10-5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. ResultsIn a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10-2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10-1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. ConclusionsRT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.

Sub-5-minute Detection of SARS-CoV-2 RNA using a Reverse Transcriptase-Free Exponential Amplification Reaction, RTF-EXPAR (preprint)

We report a rapid isothermal method for detecting SARS-CoV-2, the virus responsible for COVID-19. The procedure uses a novel reverse transcriptase-free (RTF) approach for converting RNA into DNA, which triggers a rapid amplification using the Exponential Amplification Reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection of a sample of patient SARS-CoV-2 RNA in under 5 minutes.

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins (preprint)

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Phosphorylated SARS-CoV N was shown to bind to the host 14-3-3 protein in the cytoplasm. Proteomic data indicate that seven human 14-3-3 proteins are highly abundant in human tissues vulnerable to SARS-CoV-2 infection, collectively reaching ~1.8% of all proteins in the lungs, ~1.4% in the gastrointestinal system, ~2.3% in the nervous system. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its mechanism and the specific critical phosphosites were unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein hub, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and determine the apparent KD to selected isoforms in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, located within the SR-rich region of N. The tight 14-3-3/pN association suggests it could regulate nucleocytoplasmic shuttling of N, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

S-variant SARS-CoV-2 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-QPCR (preprint)

Birmingham University Turnkey laboratory is part of the Lighthouse network responsible for testing clinical samples under the UK government ‘ Test & Trace’ scheme. Samples are analysed for the presence of SARS-CoV-2 in respiratory samples using the Thermofisher TaqPath RT-QPCR test, which is designed to co-amplify sections of three SARS-CoV-2 viral genes. Since more recent information became available regarding the presence of SARS-CoV-2 variants of concern (S-VoC), which can show a suboptimal profile in RT-QPCR tests such as the ThermoFisher TaqPath used at the majority of Lighthouse laboratories, we analysed recently published data for trends and significance of the S-gene ‘dropout’ variant. Results: showed that: the population of S-gene dropout samples had significantly lower median Ct values of ORF and N-gene targets compared to samples where S-gene was detected on a population basis, S-gene dropout samples clustered around very low Ct values for ORF and N targets linked Ct values for individual samples showed that a low Ct for ORF and N were clearly associated with an S-dropout characteristic when conservatively inferring relative viral load from Ct values, approximately 35% of S-dropout samples had high viral loads between 10 and 10,000-fold greater than 1 × 10 6 , compared to 10% of S-positive samples. This analysis suggests that patients whose samples exhibit the S-dropout profile in the TaqPath test are more likely to have high viral loads at the time of sampling. The relevance of this to epidemiological reports of fast spread of the SARS-CoV-2 in regions of the UK is discussed.

Disruption of nasal bacteria enhances protective immune responses to influenza A virus and SARS-CoV-2 infection in mice (preprint)

Gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here we demonstrate that while intranasal administration of influenza virus hemagglutinin vaccine alone was insufficient to induce the vaccine-specific antibody responses, disruption of nasal bacteria by lysozyme or addition of culturable oral bacteria from a healthy human volunteer rescued inability of the nasal bacteria to generate antibody responses to intranasally administered the split-virus vaccine. Myd88-depdnent signaling in the hematopoietic compartment was required for adjuvant activity of intranasally administered oral bacteria. In addition, we found that the oral bacteria-combined intranasal vaccine induced protective antibody response to influenza virus and SARS-CoV-2 infection. Our findings here have identified a previously unappreciated role for nasal bacteria in the induction of the virus-specific adaptive immune responses.

Diagnostic accuracy of Loop mediated isothermal amplification coupled to Nanopore sequencing for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations (preprint)

IntroductionRapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as reverse transcriptase quantitative PCR (RT-qPCR), particularly throughout the first months of the COVID-19 pandemic. We investigated the use of LamPORE, where loop mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. MethodsIn an asymptomatic prospective cohort, for three weeks in September 2020 health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 - June 2020, were similarly tested from nasopharyngeal swabs. ResultsIn the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% in both swab and saliva asymptomatic samples when compared to the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. ConclusionsLamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

Extrapolation of United Kingdom Pillar 2 Care home Covid-19 test data to ascertain effectiveness of lateral flow testing in low prevalence settings (preprint)

Lateral flow devices are quickly being implemented for use in large scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city wide screening in the city of Liverpool, and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Very little data exists comparing the performance of the UK lateral flow tests with gold standard PCR diagnostics, especially against comparable test populations such as the national Pillar 2 testing program in the United Kingdom. Here we utilise thousands of pillar 2 test data from our University of Birmingham test lab, and by extrapolation against the validate limit-of-detection of the lateral flow assay, provide a potential sensitivity for the test in a comparable low prevalence population captured in the pillar 2 program. Our data suggests the lateral flow assay should successfully capture around 85% of all PCR positive tests performed in our pillar 2 laboratory, and that a fully designed comparative study of lateral flow versus PCR testing is merited in a real life testing environment

SARS-CoV-2 seroconversion in health care workers (preprint)

Background The correlates of protection against SARS-CoV-2 and their longevity remain unclear. Studies in severely ill individuals have identified robust cellular and humoral immune responses against the virus. Asymptomatic infection with SARS-CoV-2 has also been described, but it is unknown whether this is sufficient to produce antibody responses. Methods We performed a cross-sectional study recruiting 554 health care workers from University Hospitals Birmingham NHS Foundation Trust who were at work and asymptomatic. Participants were tested for current infection with SARS-CoV-2 by nasopharyngeal swab for real-time polymerase chain reaction and for seroconversion by the measurement of anti-SARS-CoV-2 spike glycoprotein antibodies by enzyme linked immunosorbent assay. Results were interpreted in the context of previous, self-reported symptoms of illness consistent with COVID-19. Results The point prevalence of infection with SARS-CoV-2, determined by the detection of SARS-CoV-2 RNA on nasopharnygeal swab was 2.39% (n=13/544). Serum was available on 516 participants. The overall rate of seroconversion in the cohort was 24.4% (n=126/516). Individuals who had previously experienced a symptomatic illness consistent with COVID-19 had significantly greater seroconversion rates than those who had remained asymptomatic (37.5% vs 17.1%, {chi}2 =21.1034, p<0.0001). In the week preceding peak COVID-19-related mortality at UHBFT, seroconversion rates amongst those who were suffering from symptomatic illnesses peaked at 77.8%. Prior symptomatic illness generated quantitatively higher antibody responses than asymptomatic seroconversion. Seroconversion rates were highest amongst those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%) with lower rates observed in participants working in intensive care (14.8%) and emergency medicine (13.3%). Conclusions In a large cross-sectional seroprevalence study of health-care workers, we demonstrate that asymptomatic seroconversion occurs, however prior symptomatic illness is associated with quantitatively higher antibody responses. The identification that the potential for seroconversion in health-care workers can associate differentially with certain hospital departments may inform future infection control and occupational health practices.