Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

An Overview of SARS-CoV-2 Molecular Diagnostics in Europe.

The COVID-19 pandemic has led to the rapid development of a plethora of molecular diagnostic assays with real-time polymerase chain reaction (RT-PCR) at the forefront. In this review, we will discuss the history and utility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) molecular diagnostics and the associated current and future regulatory process in Europe. We will assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests currently used in Europe with a focus on as rapid molecular platforms, stand-alone RT-PCR kits, the role of low-throughput and high-throughput end-to-end testing platforms, and the rapidly evolving field of SARS-CoV-2 variant of concern identification.

Evaluation of the artus® Prep&Amp UM RT-PCR for detection of SARS-CoV-2 from nasopharyngeal swabs without prior nucleic acid eluate extraction

Here we describe a retrospective clinical evaluation of the QIAGEN artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay that detects SARS-CoV-2 RNA without the need for a nucleic acid eluate extraction procedure. Using Roche SARS-CoV-2 RT-PCR on the cobas® 8800 platform as a reference standard, a total of 225 confirmed SARS-CoV-2 positive and 320 negative nasopharyngeal swabs in viral transport media, were used to evaluate the artus® assay. Using the RT-PCR cycle threshold as a semi-quantitative marker of viral load, an assessment of over 370,000 SARS-CoV-2 RT-PCR positive results was used in the design of the reference positive specimen cohort. The viral load of all reference positive specimens used in the evaluation was a unique and accurate representation of the range and levels of SARS-CoV-2 positivity observed over a 13-month period of the COVID-19 pandemic. The artus® RT-PCR detects the presence of SARS-CoV-2 RNA, an internal control, and the human RNase P gene to ensure specimen quality. The diagnostic sensitivity of artus® was 92.89% with a specificity of 100%. To assess the analytical sensitivity, a limit of detection was performed using the 1st WHO NIBSC SARS-CoV-2 international standard, recording a 95% LOD of 1.1 × 103 IU/ml. The total invalid rate of specimens was 7.34% due to a lack of detectable RNase P (Ct >35). The artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay is a new rapid RT-PCR assay, which may be considered to produce acceptable levels of diagnostic sensitivity and specificity whilst potentially halving the laboratory processing time.

Evaluation of a novel direct RT-LAMP assay for the detection of SARS-CoV-2 from saliva samples in asymptomatic individuals.

Large scale screening of health care workers and the general population for asymptomatic COVID-19 infection requires modalities that are amenable to testing at scale while retaining acceptable levels of sensitivity and specificity. This study evaluated a novel COVID-19 Direct-RT LAMP assay using saliva samples in asymptomatic individuals by comparison to RT-PCR. Additional studies were performed using VTM collected from routine diagnostic testing. Analytical sensitivity was determined for Direct RT-LAMP assay using the WHO International Standard. Finally, quantified results from RT-PCR testing of 9177 nose and throat swabs obtained from routine diagnostic testing were used to estimate the sensitivity of Direct RT-LAMP using the limit of detection curve obtained from the analytical sensitivity data. Results from saliva testing demonstrated a sensitivity of 40.91% and a specificity of 100% for Direct RT-LAMP. The sensitivity and specificity for nose and throat swabs were 44.85% and 100% respectively. The 95% limit of detection (LOD) for Direct RT-LAMP was log 7.13 IU/ml (95% 6.9-7.5). The estimated sensitivity for Direct-RT LAMP based on the results of 9117 nose and throat swabs was 34% and 45% for saliva and VTM respectively. The overall diagnostic sensitivity of Direct RT-LAMP was low compared to RT-PCR. Testing of nose and throat swabs and estimating the sensitivity based on a large cohort of clinical samples demonstrated similar results. This study highlights the importance of utilising the prospective collection of samples from the intended target population in the assessment of diagnostic sensitivity.

Use of dried blood spot samples for SARS-CoV-2 antibody detection using the Roche Elecsys ® high throughput immunoassay.

Dried blood spot samples (DBS) provide an alternative sample type to venous blood samples for antibody testing. DBS are used by NHS for diagnosing Hepatitis C and by Public Health England for large scale HIV and Hepatitis C serosurveillance; the applicability of DBS based approaches for SARS-CoV-2 antibody detection is uncertain. The study aimed to compare antibody detection in DBS eluates using the Roche Elecsys ® immunoassay with antibody detection in paired plasma samples, using the same assay. The study was in one Police and one Fire & Rescue facility in England; it comprised of 195 participants within a larger sample COVID-19 serodiagnostics study of keyworkers, EDSAB-HOME. Outcome measures were sensitivity and specificity of DBS (the index test) relative to plasma (the reference test), at an experimental cut-off; quality of DBS sample collected; estimates of relative sensitivity of DBS vs. plasma immunoassay in a larger population. 18/195 (9.2%) participants tested positive using plasma samples. DBS sample quality varied markedly by phlebotomist, and low sample volume significantly reduced immunoassay signals. Using an experimental cut-off, sensitivity and specificity of DBS were 89.0% (95% CI 67.2, 96.9%) and 100.0% (95% CI 97.9, 100%) respectively compared with using plasma. The limit of detection for DBS is about 30 times higher than for plasma. DBS use for SARS-CoV-2 serology, though feasible, is insensitive relative to immunoassays on plasma. Sample quality impacts on assay performance. Alternatives, including the collection of capillary blood samples, should be considered for screening programs.

Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations.

OBJECTIVES: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as 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 quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 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. METHODS: In an asymptomatic prospective cohort, for 3 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 to June 2020 were similarly tested from nasopharyngeal swabs. RESULTS: In the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. CONCLUSIONS: LamPORE 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.

Real-world SARS CoV-2 testing in Northern England during the first wave of the COVID-19 pandemic.

OBJECTIVES: SARS-CoV-2 emerged in South Asia in 2019 and has resulted in a global pandemic. Public Health England (PHE) Manchester rapidly escalated testing for SARS-CoV-2 in the highest COVID-19 incidence location in England. The results of the PHE Manchester SARS-CoV-2 surveillance during the first wave are presented. METHODS: Retrospective data were collected for patients fitting the PHE SARS-CoV-2 case definition from 11th February to 31st August 2020. Respiratory tract, tissue, faecal, fluid and cerebrospinal (CSF) samples were tested for SARS-CoV-2 by a semi-quantitative real-time reverse-transcription PCR. RESULTS: Of the 204,083 tests for SARS-CoV-2, 18,011 were positive demonstrating a positivity of 8.90%. Highest positivity was in nasal swabs (20.99%) followed by broncheo-alveolar lavage samples (12.50%). None of the faecal, fluid or CSF samples received were positive for SARS-CoV-2. CONCLUSIONS: There was a high incidence of SARS-CoV-2 patients in the North-West of England during the first UK wave of the Covid-19 pandemic. Highest positivity rate was in nasal specimens suggesting this is the optimum sample type within this dataset for detecting SARS-CoV-2. Further studies are warranted to assess the utility of testing faecal, fluid and CSF samples. Rapid escalation of testing via multiple platforms was required to ensure prompt diagnosis and isolate infected cases to reduce transmission of the virus.

Genomic and healthcare dynamics of nosocomial SARS-CoV-2 transmission.

Understanding the effectiveness of infection control methods in reducing and preventing SARS-CoV-2 transmission in healthcare settings is of high importance. We sequenced SARS-CoV-2 genomes for patients and healthcare workers (HCWs) across multiple geographically distinct UK hospitals, obtaining 173 high-quality SARS-CoV-2 genomes. We integrated patient movement and staff location data into the analysis of viral genome data to understand spatial and temporal dynamics of SARS-CoV-2 transmission. We identified eight patient contact clusters (PCC) with significantly increased similarity in genomic variants compared to non-clustered samples. Incorporation of HCW location further increased the number of individuals within PCCs and identified additional links in SARS-CoV-2 transmission pathways. Patients within PCCs carried viruses more genetically identical to HCWs in the same ward location. SARS-CoV-2 genome sequencing integrated with patient and HCW movement data increases identification of outbreak clusters. This dynamic approach can support infection control management strategies within the healthcare setting.

A Prospective Study to Identify Rates of SARS-CoV-2 Virus in the Peritoneum and Lower Genital Tract of Patients Having Surgery: An Observational Study.

STUDY OBJECTIVE: The risks to surgeons of carrying out aerosol-generating procedures during the coronavirus disease 2019 (COVID-19) pandemic are unknown. To start to define these risks, in a systematic manner, we investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in the abdominal fluid and lower genital tract of patients undergoing surgery. DESIGN: Prospective cross-sectional observational study. SETTING: Single, large United Kingdom hospital. PATIENTS: Total of 113 patients undergoing abdominal surgery or instrumentation of the lower genital tract. INTERVENTIONS: We took COVID-19 swabs from the peritoneal cavity and from the vagina from all eligible patients. Results were stratified by preoperative COVID-19 status. MEASUREMENTS AND MAIN RESULTS: In patients who were presumed COVID-19 negative at the time of surgery, SARS-CoV-2 virus RNA was detected in 0 of 102 peritoneal samples and 0 of 98 vaginal samples. Both cohorts included 4 patients who were antibody positive but nasopharyngeal swab test negative at the time of surgery. Peritoneal and vaginal swabs were also negative in 1 patient who had a positive nasopharyngeal swab immediately before surgery. CONCLUSION: The presence of SARS-CoV-2 RNA in the abdominal fluid or lower genital tract of presumed negative patients is nil or extremely low. These data will inform surgeons of the risks of restarting laparoscopic surgery at a time when COVID-19 is endemic in the population.