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Biochem J ; 479(8): 901-920, 2022 04 29.
Article in English | MEDLINE | ID: covidwho-1774010


Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

COVID-19 , Metal Nanoparticles , Antiviral Agents , COVID-19/diagnosis , Colorimetry , Coronavirus 3C Proteases , Gold , HEK293 Cells , Humans , Peptide Hydrolases , Protease Inhibitors , SARS-CoV-2
Sci Rep ; 12(1): 4599, 2022 03 29.
Article in English | MEDLINE | ID: covidwho-1768844


Dehydration of the upper airways increases risks of respiratory diseases from COVID-19 to asthma and COPD. We find in human volunteer studies involving 464 human subjects in Germany, the US, and India that respiratory droplet generation increases by up to 4 orders of magnitude in dehydration-associated states of advanced age (n = 357), elevated BMI-age (n = 148), strenuous exercise (n = 20) and SARS-CoV-2 infection (n = 87), and falls with hydration of the nose, larynx and trachea by calcium-rich hypertonic salts. We also find in a protocol of exercise-induced airway dehydration that hydration of the airways by calcium-rich salts increases oxygenation relative to a non-treatment control (P < 0.05). In a random control study of COVID-19 positive subjects (n = 40), thrice-a-day delivery of the calcium-rich hypertonic salts (active) suppressed respiratory droplet generation by 51% ± 11% and increased oxygen saturation over three days of treatment by 48.08% ± 9.61% (P < 0.001), while no changes were observed in the nasal-saline control group. Self-reported symptoms significantly declined in the active group and did not decline in the control group. Hydration of the upper airways appears promising as a non-drug approach for reducing risks of respiratory diseases such as COVID-19.

COVID-19 , Larynx , Exercise , Humans , SARS-CoV-2 , Trachea
J Infect ; 82(6): 253-259, 2021 06.
Article in English | MEDLINE | ID: covidwho-1152506


BACKGROUND: Human to human transmission of SARS-CoV-2 is driven by the respiratory route but little is known about the pattern and quantity of virus output from exhaled breath. We have previously shown that face-mask sampling (FMS) can detect exhaled tubercle bacilli and have adapted its use to quantify exhaled SARS-CoV-2 RNA in patients admitted to hospital with Coronavirus Disease-2019 (COVID-19). METHODS: Between May and December 2020, we took two concomitant FMS and nasopharyngeal samples (NPS) over two days, starting within 24 h of a routine virus positive NPS in patients hospitalised with COVID-19, at University Hospitals of Leicester NHS Trust, UK. Participants were asked to wear a modified duckbilled facemask for 30 min, followed by a nasopharyngeal swab. Demographic, clinical, and radiological data, as well as International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) mortality and deterioration scores were obtained. Exposed masks were processed by removal, dissolution and analysis of sampling matrix strips fixed within the mask by RT-qPCR. Viral genome copy numbers were determined and results classified as Negative; Low: ≤999 copies; Medium: 1000-99,999 copies and High ≥ 100,000 copies per strip for FMS or per 100 µl for NPS. RESULTS: 102 FMS and NPS were collected from 66 routinely positive patients; median age: 61 (IQR 49 - 77), of which FMS was positive in 38% of individuals and concomitant NPS was positive in 50%. Positive FMS viral loads varied over five orders of magnitude (<10-3.3 x 106 genome copies/strip); 21 (32%) patients were asymptomatic at the time of sampling. High FMS viral load was associated with respiratory symptoms at time of sampling and shorter interval between sampling and symptom onset (FMS High: median (IQR) 2 days (2-3) vs FMS Negative: 7 days (7-10), p = 0.002). On multivariable linear regression analysis, higher FMS viral loads were associated with higher ISARIC mortality (Medium FMS vs Negative FMS gave an adjusted coefficient of 15.7, 95% CI 3.7-27.7, p = 0.01) and deterioration scores (High FMS vs Negative FMS gave an adjusted coefficient of 37.6, 95% CI 14.0 to 61.3, p = 0.002), while NPS viral loads showed no significant association. CONCLUSION: We demonstrate a simple and effective method for detecting and quantifying exhaled SARS-CoV-2 in hospitalised patients with COVID-19. Higher FMS viral loads were more likely to be associated with developing severe disease compared to NPS viral loads. Similar to NPS, FMS viral load was highest in early disease and in those with active respiratory symptoms, highlighting the potential role of FMS in understanding infectivity.

COVID-19 , SARS-CoV-2 , Humans , Masks , Middle Aged , RNA, Viral , Viral Load