Bayesian Statistical Analysis for Bacterial Detection in Pulmonary Endomicroscopic Fluorescence Lifetime Imaging.

Pneumonia, a respiratory disease often caused by bacterial infection in the distal lung, requires rapid and accurate identification, especially in settings such as critical care. Initiating or de-escalating antimicrobials should ideally be guided by the quantification of pathogenic bacteria for effective treatment. Optical endomicroscopy is an emerging technology with the potential to expedite bacterial detection in the distal lung by enabling in vivo and in situ optical tissue characterisation. With advancements in detector technology, optical endomicroscopy can utilize fluorescence lifetime imaging (FLIM) to help detect events that were previously challenging or impossible to identify using fluorescence intensity imaging. In this paper, we propose an iterative Bayesian approach for bacterial detection in FLIM. We model the FLIM image as a linear combination of background intensity, Gaussian noise, and additive outliers (labelled bacteria). While previous bacteria detection methods model anomalous pixels as bacteria, here the FLIM outliers are modelled as circularly symmetric Gaussian-shaped objects, based on their discrete shape observed through visual analysis and the physical nature of the imaging modality. A Hierarchical Bayesian model is used to solve the bacterial detection problem where prior distributions are assigned to unknown parameters. A Metropolis-Hastings within Gibbs sampler draws samples from the posterior distribution. The proposed method's detection performance is initially measured using synthetic images, and shows significant improvement over existing approaches. Further analysis is conducted on real optical endomicroscopy FLIM images annotated by trained personnel. The experiments show the proposed approach outperforms existing methods by a margin of +16.85% ( F1 ) for detection accuracy.

A Novel Fit-Flexible Fluorescence Soft Imager: Tri-Sensing of Intensity, Fall-Time, and Life Profile.

Time-resolved fluorescence imaging techniques, like confocal fluorescence lifetime imaging microscopy, are powerful photonic instrumentation tools of modern science with diverse applications, including: biology, medicine, and chemistry. However, complexities of the systems, both at specimen and device levels, cause difficulties in quantifying soft biomarkers. To address the problems, we first aim to understand and model the underlying photophysics of fluorescence decay curves. For this purpose, we provide a set of mathematical functions, called "life models", fittable with the real temporal recordings of histogram of photon counts. For each model, an equivalent electrical circuit, called a "life circuit", is derived for explaining the whole process. In confocal endomicroscopy, the components of excitation laser, specimen, and fluorescence-emission signal as the histogram of photon counts are modelled by a power source, network of resistor-inductor-capacitor circuitry, and multimetre, respectively. We then design a novel pixel-level temporal classification algorithm, called a "fit-flexible approach", where qualities of "intensity", "fall-time", and "life profile" are identified for each point. A model selection mechanism is used at each pixel to flexibly choose the best representative life model based on a proposed Misfit-percent metric. A two-dimensional arrangement of the quantified information detects some kind of structural information. This approach showed a potential of separating microbeads from lung tissue, distinguishing the tri-sensing from conventional methods. We alleviated by 7% the error of the Misfit-percent for recovering the histograms on real samples than the best state-of-the-art competitor. Codes are available online.

Phase 2 randomised placebo-controlled trial of spironolactone and dexamethasone versus dexamethasone in COVID-19 hospitalised patients in Delhi.

BACKGROUND: In this phase 2 randomised placebo-controlled clinical trial in patients with COVID-19, we hypothesised that blocking mineralocorticoid receptors using a combination of dexamethasone to suppress cortisol secretion and spironolactone is safe and may reduce illness severity. METHODS: Hospitalised patients with confirmed COVID-19 were randomly allocated to low dose oral spironolactone (50 mg day 1, then 25 mg once daily for 21 days) or standard of care in a 2:1 ratio. Both groups received dexamethasone 6 mg daily for 10 days. Group allocation was blinded to the patient and research team. Primary outcomes were time to recovery, defined as the number of days until patients achieved WHO Ordinal Scale (OS) category ≤ 3, and the effect of spironolactone on aldosterone, D-dimer, angiotensin II and Von Willebrand Factor (VWF). RESULTS: One hundred twenty patients with PCR confirmed COVID were recruited in Delhi from 01 February to 30 April 2021. 74 were randomly assigned to spironolactone and dexamethasone (SpiroDex), and 46 to dexamethasone alone (Dex). There was no significant difference in the time to recovery between SpiroDex and Dex groups (SpiroDex median 4.5 days, Dex median 5.5 days, p = 0.055). SpiroDex patients had significantly lower D-dimer levels on days 4 and 7 (day 7 mean D-dimer: SpiroDex 1.15 µg/mL, Dex 3.15 µg/mL, p = 0.0004) and aldosterone at day 7 (SpiroDex 6.8 ng/dL, Dex 14.52 ng/dL, p = 0.0075). There was no difference in VWF or angiotensin II levels between groups. For secondary outcomes, SpiroDex patients had a significantly greater number of oxygen free days and reached oxygen freedom sooner than the Dex group. Cough scores were no different during the acute illness, however the SpiroDex group had lower scores at day 28. There was no difference in corticosteroid levels between groups. There was no increase in adverse events in patients receiving SpiroDex. CONCLUSION: Low dose oral spironolactone in addition to dexamethasone was safe and reduced D-dimer and aldosterone. Time to recovery was not significantly reduced. Phase 3 randomised controlled trials with spironolactone and dexamethasone should be considered. TRIAL REGISTRATION: The trial was registered on the Clinical Trials Registry of India TRI: CTRI/2021/03/031721, reference: REF/2021/03/041472. Registered on 04/03/2021.

Specific in situ immuno-imaging of pulmonary-resident memory lymphocytes in human lungs.

Introduction: Pulmonary-resident memory T cells (TRM) and B cells (BRM) orchestrate protective immunity to reinfection with respiratory pathogens. Developing methods for the in situ detection of these populations would benefit both research and clinical settings. Methods: To address this need, we developed a novel in situ immunolabelling approach combined with clinic-ready fibre-based optical endomicroscopy (OEM) to detect canonical markers of lymphocyte tissue residency in situ in human lungs undergoing ex vivo lung ventilation (EVLV). Results: Initially, cells from human lung digests (confirmed to contain TRM/BRM populations using flow cytometry) were stained with CD69 and CD103/CD20 fluorescent antibodies and imaged in vitro using KronoScan, demonstrating it's ability to detect antibody labelled cells. We next instilled these pre-labelled cells into human lungs undergoing EVLV and confirmed they could still be visualised using both fluorescence intensity and lifetime imaging against background lung architecture. Finally, we instilled fluorescent CD69 and CD103/CD20 antibodies directly into the lung and were able to detect TRM/BRM following in situ labelling within seconds of direct intra-alveolar delivery of microdoses of fluorescently labelled antibodies. Discussion: In situ, no wash, immunolabelling with intra-alveolar OEM imaging is a novel methodology with the potential to expand the experimental utility of EVLV and pre-clinical models.

An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics.

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Evaluation of new or repurposed treatments for COVID-19: protocol for the phase Ib/IIa DEFINE trial platform.

INTRODUCTION: COVID-19 is a new viral-induced pneumonia caused by infection with a novel coronavirus, SARS-CoV-2. At present, there are few proven effective treatments. This early-phase experimental medicine protocol describes an overarching and adaptive trial designed to provide safety data in patients with COVID-19, pharmacokinetic (PK)/pharmacodynamic (PD) information and exploratory biological surrogates of efficacy, which may support further development and deployment of candidate therapies in larger scale trials of patients positive for COVID-19. METHODS AND ANALYSIS: Define is an ongoing exploratory multicentre-platform, open-label, randomised study. Patients positive for COVID-19 will be recruited from the following cohorts: (a) community cases; (b) hospitalised patients with evidence of COVID-19 pneumonitis; and (c) hospitalised patients requiring assisted ventilation. The cohort recruited from will be dependent on the experimental therapy, its route of administration and mechanism of action. Randomisation will be computer generated in a 1:1:n ratio. Twenty patients will be recruited per arm for the initial two arms. This is permitted to change as per the experimental therapy. The primary statistical analyses are concerned with the safety of candidate agents as add-on therapy to standard of care in patients with COVID-19. Secondary analysis will assess the following variables during treatment period: (1) the response of key exploratory biomarkers; (2) change in WHO ordinal scale and National Early Warning Score 2 (NEWS2) score; (3) oxygen requirements; (4) viral load; (5) duration of hospital stay; (6) PK/PD; and (7) changes in key coagulation pathways. ETHICS AND DISSEMINATION: The Define trial platform and its initial two treatment and standard of care arms have received a favourable ethical opinion from Scotland A Research Ethics Committee (REC) (20/SS/0066), notice of acceptance from The Medicines and Healthcare Products Regulatory Agency (MHRA) (EudraCT 2020-002230-32) and approval from the relevant National Health Service (NHS) Research and Development (R&D) departments (NHS Lothian and NHS Greater Glasgow and Clyde). Appropriate processes are in place in order to be able to consent adults with and without capacity while following the necessary COVID-19 safe procedures. Patients without capacity could be recruited via a legal representative. Witnessed electronic consent of participants or their legal representatives following consent discussions was established. The results of each study arm will be submitted for publication in a peer-reviewed journal as soon as the treatment arm has finished recruitment, data input is complete and any outstanding patient safety follow-ups have been completed. Depending on the results of these or future arms, data will be shared with larger clinical trial networks, including the Randomised Evaluation of COVID-19 Therapy trial (RECOVERY), and to other partners for rapid roll-out in larger patient cohorts. TRIAL REGISTRATION NUMBER: ISRCTN14212905, NCT04473053.