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1.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.11.05.21265890

ABSTRACT

Respiratory infections are the major cause of death from infectious disease worldwide. The clinical presentation of many respiratory viruses is indistinguishable; therefore, diagnostic approaches that can identify multiple pathogens are essential for patient management. We aimed to address this challenge with 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 SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus, influenza, and parainfluenza. Our nanobait could be reprogrammed to discriminate viral variants, and we identified several key SARS-CoV-2 variants with single-nucleotide resolution. We increased assay specificity with bespoke nanobait that could identify numerous short RNA targets in the same viral sample in a complex background of the human transcriptome. Notably, we found that the sequence position in the viral RNA secondary structure is critical for nanobait design. Lastly, we show that nanobait could discriminate between samples extracted from oropharyngeal swabs from negative and positive SARS-CoV-2 patients using programmable target cleavage 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.


Subject(s)
28582 , 3660 , 12561
2.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.09.16.21263684

ABSTRACT

Summary Background The COVID-19 pandemic has overwhelmed the respiratory isolation capacity in hospitals; many wards lacking high-frequency air changes have been repurposed for managing patients infected with SARS-CoV-2 requiring either standard or intensive care. Hospital-acquired COVID-19 is a recognised problem amongst both patients and staff, with growing evidence for the relevance of airborne transmission. This study examined the effect of air filtration and ultra-violet (UV) light sterilisation on detectable airborne SARS-CoV-2 and other microbial bioaerosols. Methods We conducted a crossover study of portable air filtration and sterilisation devices in a repurposed ‘surge’ COVID ward and ‘surge’ ICU. National Institute for Occupational Safety and Health (NIOSH) cyclonic aerosol samplers and PCR assays were used to detect the presence of airborne SARS-CoV-2 and other microbial bioaerosol with and without air/UV filtration. Results Airborne SARS-CoV-2 was detected in the ward on all five days before activation of air/UV filtration, but on none of the five days when the air/UV filter was operational; SARS-CoV-2 was again detected on four out of five days when the filter was off. Airborne SARS-CoV-2 was infrequently detected in the ICU. Filtration significantly reduced the burden of other microbial bioaerosols in both the ward (48 pathogens detected before filtration, two after, p =0.05) and the ICU (45 pathogens detected before filtration, five after p =0.05). Conclusions These data demonstrate the feasibility of removing SARS-CoV-2 from the air of repurposed ‘surge’ wards and suggest that air filtration devices may help reduce the risk of hospital-acquired SARS-CoV-2. Funding Wellcome Trust, MRC, NIHR


Subject(s)
59585
3.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.06.26.20139873

ABSTRACT

Background Pandemic 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). Objectives To 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. Methods In 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. Results We 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. Conclusion COVID-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.


Subject(s)
11443 , 59585 , 37050 , 55222 , 52093
4.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.06.02.20118489

ABSTRACT

Background The diagnosis of infectious diseases has been hampered by reliance on microbial culture. Cultures take several days to return a result and organisms frequently fail to grow. In critically ill patients this leads to the use of empiric, broad-spectrum antimicrobials and mitigates against stewardship. Methods Single ICU observational cohort study with contemporaneous comparator group. We developed and implemented a TaqMan array card (TAC) covering 52 respiratory pathogens in ventilated patients undergoing bronchoscopic investigation for suspected pneumonia. The time to result was compared against conventional culture, and sensitivity compared to conventional microbiology and metagenomic sequencing. We observed the clinician decisions in response to array results, comparing antibiotic free days (AFD) between the study cohort and comparator group. Findings 95 patients were enrolled with 71 forming the comparator group. TAC returned results 61 hours (IQR 42-90) faster than culture. The test had an overall sensitivity of 93% (95% CI 88-97%) compared to a combined standard of conventional culture and metagenomic sequencing, with 100% sensitivity for most individual organisms. In 54% of cases the TAC results altered clinical management, with 62% of changes leading to de-escalation, 30% to an increase in spectrum, and investigations for alternative diagnoses in 9%. There was a significant difference in the distribution of AFDs with more AFDs in the TAC group (p=0.02). Interpretation Implementation of a customised syndromic diagnostic for pneumonia led to faster results, with high sensitivity and measurable impact on clinical decision making. Funding Addenbrookes Charitable Trust, Wellcome Trust and Cambridge NIHR BRC


Subject(s)
28582 , 11443 , 22442 , 29398
5.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.05.09.20082909

ABSTRACT

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.


Subject(s)
59585 , 386
6.
biorxiv; 2020.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2020.04.14.041319

ABSTRACT

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.


Subject(s)
59585
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