Simultaneous identification of viruses and SARS-CoV-2 variants with programmable DNA nanobait

Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid read-out via nanopore sensing. Here, we show this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants, as we demonstrated for several key SARS-CoV-2 variants with single-nucleotide resolution. Lastly, we show that nanobait discriminates between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients without pre-amplification. Our system allows for multiplexed identification of native RNA molecules, providing a new scalable approach for diagnostics of multiple respiratory viruses in a single assay.

Secondary pneumonia in critically ill ventilated patients with COVID-19

BackgroundPandemic COVID-19 caused by the coronavirus SARS-CoV-2 has a high incidence of patients with severe acute respiratory syndrome (SARS). Many of these patients require admission to an intensive care unit (ICU) for invasive artificial ventilation and are at significant risk of developing a secondary, ventilator-associated pneumonia (VAP). ObjectivesTo study the incidence of VAP, as well as differences in secondary infections, and bacterial lung microbiome composition of ventilated COVID-19 and non-COVID-19 patients. MethodsIn this prospective observational study, we compared the incidence of VAP and secondary infections using a combination of a TaqMan multi-pathogen array and microbial culture. In addition, we determined the lung microbime composition using 16S RNA analyisis. The study involved eighteen COVID-19 and seven non-COVID-19 patients receiving invasive ventilation in three ICUs located in a single University teaching hospital between April 13th 2020 and May 7th 2020. ResultsWe observed a higher percentage of confirmed VAP in COVID-19 patients. However, there was no statistical difference in the detected organisms or pulmonary microbiome when compared to non-COVID-19 patients. ConclusionCOVID-19 makes people more susceptible to developing VAP, partly but not entirely due to the increased duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the array of secondary infections observed are similar to that seen in critically ill patients ventilated for other reasons.

Development and implementation of a customised rapid syndromic diagnostic test for severe pneumonia

BackgroundMicrobial cultures for the diagnosis of pneumonia take several days to return a result, and are frequently negative, compromising antimicrobial stewardship. The objective of this study was to establish the performance of a syndromic molecular diagnostic approach, using a custom TaqMan array card (TAC) covering 52 respiratory pathogens, and assess its impact on antimicrobial prescribing. MethodsThe TAC was validated against a retrospective multi-centre cohort of broncho-alveolar lavage samples. The TAC was assessed prospectively in patients undergoing investigation for suspected pneumonia, with a comparator cohort formed of patients investigated when the TAC laboratory team were unavailable. Co-primary outcomes were sensitivity compared to conventional microbiology and, for the prospective study, time to result. Metagenomic sequencing was performed to validate findings in prospective samples. Antibiotic free days (AFD) were compared between the study cohort and comparator group. Results128 stored samples were tested, with sensitivity of 97% (95% CI 88-100%). Prospectively 95 patients were tested by TAC, with 71 forming the comparator group. TAC returned results 51 hours (IQR 41-69 hours) faster than culture and with sensitivity of 92% (95% CI 83-98%) compared to conventional microbiology. 94% of organisms identified by sequencing were detected by TAC. There was a significant difference in the distribution of AFDs with more AFDs in the TAC group (p=0.02). TAC group were more likely to experience antimicrobial de-escalation (OR 2.9 (95%1.5-5.5). ConclusionsImplementation of a syndromic molecular diagnostic approach to pneumonia led to faster results, with high sensitivity and impact on antibiotic prescribing. Trial registrationThe prospective study was registered with clinicaltrials.gov NCT03996330

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3-week period (April 2020), 1,032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19) >7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B{middle dot}1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff. Appendix: The CITIID-NIHR COVID-19 BioResource CollaborationO_ST_ABSPrincipal InvestigatorsC_ST_ABSStephen Baker, John Bradley, Gordon Dougan, Ian Goodfellow, Ravi Gupta, Paul J. Lehner, Paul A. Lyons, Nicholas J. Matheson, Kenneth G.C. Smith, M. Estee Torok, Mark Toshner, Michael P. Weekes Infectious Diseases DepartmentNicholas K. Jones, Lucy Rivett, Matthew Routledge, Dominic Sparkes, Ben Warne SARS-CoV-2 testing teamJosefin Bartholdson Scott, Claire Cormie, Sally Forrest, Harmeet Gill, Iain Kean, Mailis Maes, Joana Pereira-Dias, Nicola Reynolds, Sushmita Sridhar, Michelle Wantoch, Jamie Young COG-UK Cambridge Sequencing TeamSarah Caddy, Laura Caller, Theresa Feltwell, Grant Hall, William Hamilton, Myra Hosmillo, Charlotte Houldcroft, Aminu Jahun, Fahad Khokhar, Luke Meredith, Anna Yakovleva NIHR BioResourceHelen Butcher, Daniela Caputo, Debra Clapham-Riley, Helen Dolling, Anita Furlong, Barbara Graves, Emma Le Gresley, Nathalie Kingston, Sofia Papadia, Hannah Stark, Kathleen E. Stirrups, Jennifer Webster Research nursesJoanna Calder, Julie Harris, Sarah Hewitt, Jane Kennet, Anne Meadows, Rebecca Rastall, Criona O,Brien, Jo Price, Cherry Publico, Jane Rowlands, Valentina Ruffolo, Hugo Tordesillas NIHR Cambridge Clinical Research FacilityKaren Brookes, Laura Canna, Isabel Cruz, Katie Dempsey, Anne Elmer, Naidine Escoffery, Stewart Fuller, Heather Jones, Carla Ribeiro, Caroline Saunders, Angela Wright Cambridge Cancer Trial CentreRutendo Nyagumbo, Anne Roberts Clinical Research Network EasternAshlea Bucke, Simone Hargreaves, Danielle Johnson, Aileen Narcorda, Debbie Read, Christian Sparke, Lucy Warboys Administrative staff, CUHKirsty Lagadu, Lenette Mactavous CUH NHS Foundation TrustTim Gould, Tim Raine, Ashley Shaw Cambridge Cancer Trials CentreClaire Mather, Nicola Ramenatte, Anne-Laure Vallier Legal/EthicsMary Kasanicki CUH Improvement and Transformation TeamPenelope-Jane Eames, Chris McNicholas, Lisa Thake Clinical Microbiology & Public Health Laboratory (PHE): Neil Bartholomew, Nick Brown, Martin Curran, Surendra Parmar, Hongyi Zhang Occupational HealthAilsa Bowring, Mark Ferris, Geraldine Martell, Natalie Quinnell, Giles Wright, Jo Wright Health and SafetyHelen Murphy Department of Medicine Sample LogisticsBenjamin J. Dunmore, Ekaterina Legchenko, Stefan Graf, Christopher Huang, Josh Hodgson, Kelvin Hunter, Jennifer Martin, Federica Mescia, Ciara ODonnell, Linda Pointon, Joy Shih, Rachel Sutcliffe, Tobias Tilly, Zhen Tong, Carmen Treacy, Jennifer Wood Department of Medicine Sample Processing and Acquisition: Laura Bergamaschi, Ariana Betancourt, Georgie Bowyer, Aloka De Sa, Maddie Epping, Andrew Hinch, Oisin Huhn, Isobel Jarvis, Daniel Lewis, Joe Marsden, Simon McCallum, Francescsa Nice, Ommar Omarjee, Marianne Perera, Nika Romashova, Mateusz Strezlecki, Natalia Savoinykh Yarkoni, Lori Turner Epic team/other computing supportBarrie Bailey, Afzal Chaudhry, Rachel Doughton, Chris Workman Statistics/modellingRichard J. Samworth, Caroline Trotter

A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities

The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 (CL2) laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the methods for a quantitative real-time reverse transcriptase PCR (qRT-PCR) detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.