Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Colourimetric sensor for monitoring ammonia-generating infectious bacteria

A simple, inexpensive bromophenol blue (BPB), colourimetric ammonia (NH3) sensor is used to monitor the growth of different wound associated bacteria in three different, simple wound models which utilise a typical, readily available, commercial occlusive wound dressing. The 25 % colour change point of the sensor is ca. 0.0032 %NH3 and its 90 % response and recovery times are, 8 and 230 s, respectively. Using P. stuartii as the test species, in a model wound with an occlusive dressing, the increase in the sensor’s colour up to the 25 % point correlates with that of the exponential growth of the bacterial species (units: CFU/mL) and a simple mathematical model is used to show that a linear relationship should exist between log(CFU/mL) of the initial inoculum and the time it takes for the sensor to reach its 25 % colour change point, the threshold time, TT. This prediction is confirmed using the NH3 producing bacteria, E. coli, E. cloacae, P. stuartii, and K. aerogenes using three different wound models, namely ones in which the ‘wound’ was a non-urea containing agar plug, a urea-containing agar plug and a damaged porcine skin model, respectively. These results suggest that the NH3 sensor has promise as a non-invasive wound monitoring indicator and the additional work required to progress the sensor in this role is discussed briefly.<br/

Ammonia colourimetric indicator for measuring urease and ureolytic bacteria concentrations

An effectively irreversible colourimetric ammonia (NH3) indicator is used to develop two new methods for determining the concentrations of, (i) urease [urease], and (ii) ureolytic bacteria under aerobic and anaerobic conditions. In both methods digital colour analysis (DCA) of a photographic image of the indicator is used to provide a value for the apparent absorbance, A′, which reflects the level of NH3 present. In (i) the indicator is placed in a solution containing urea and the urease sample under test, and photographed as a function of incubation time, t. DCA of the photographs yields an A′ vs t plot, from which a value for the time taken for the indicator to reach its half-way colour changing point, t50, is determined, the reciprocal of which is directly proportional to [urease]. In (ii), the indicator is placed in a Falcon™ tube containing a urea-based growth medium inoculated with a ureolytic bacterium of known concentration (units: CFU/mL), and photographed as a function of t. The resulting ‘S’ shaped A′ vs t profile is used to determine a value of the half-way colour change, the threshold time, TT, which is shown to be directly proportional to log(CFU/mL) over the range 101–108 CFU/mL for ureolytic bacteria such as P. stuartii and P. mirabilis, even in the presence of a non-ureolytic bacterial species, such as E. coli. The latter novel NH3 indicator based micro-respirometry method for measuring TVC (NH3 μR-TVC) works under anaerobic and aerobic conditions.<br/

Simple, reusable, solid-state system for measuring total (aerobic) viable count, TVC, using the micro-respirometry method (μRM)

Liquid micro-respirometry, Liq-μRM, is a popular, more rapid alternative method for measuring Total Viable Count of aerobes, i.e., TVC, units: colony forming units (CFU)/mL, to the traditional, time-consuming, plate counting method. Liq-μRM is based on monitoring the consumption of dissolved O2 in a liquid growth medium as a function of incubation time. However, in Liq-μRM, the O2 indicator is difficult to make, non-reusable and not well suited for use in well-plates, due to agitation issues. In this work a new, Solid-State micro-respirometry method, i.e., SS-μRM, is described based on a solid, rather than liquid, growth medium, in which the consumption of O2 in the headspace is monitored, using a 3D printed O2 indicator set in the lid of a small Petri dish or well plate containing the solid growth medium. The performance of the SS-μRM in a small Petri dish is compared with that of a commercial Liq-μRM system, using Escherichia coli, i.e., E. coli, as the test bacterium over the range 101 – 108 CFU/mL and demonstrated to be equivalent. The same system is used to measure the TVC of Pseudomonas aeruginosa, P. aeruginosa, and Enterobacter cloacae, E. cloacae, over the range 101 – 108 CFU/mL. When used to measure the TVC of E. coli, in a well plate, SS-μRM is shown to be insensitive to agitation, in contrast to Liq-μRM. The ability of the SS-μRM to address the main concerns regarding Liq-μRM are discussed.<br/

Early wound infection monitoring via headspace O2 micro-respirometry

A luminescence based, inexpensive, 3D printed O2 indicator is incorporated into a commercial, clear, occlusive wound dressing, which allows the %O2 in the headspace above a simulated wound to be monitored. Two wound models are used to evaluate this micro-respirometry-based system for monitoring wound infection namely, a simple ‘agar plug’ model and a wounded porcine skin model. Inoculation of either wound model with E. coli, E. cloacae, or A. baumannii, produces the typical ‘S’-shaped, τ vs incubation time, t, profiles, associated with micro-respirometry, due to the decrease in %O2 in the headspace above the wound. A threshold value for the lifetime, τTT, of 21.1 μs, is identified at which the bacterial load is equal to the critical colonization threshold, CCT, ca. 106 colony forming units, CFU/mL, above which infection is highly likely. The agar plug wound model/O2 indicator combination is used to identify when the CCT is reached for a wide range of inoculant concentrations, spanning the range 108–101 CFU/mL, for all three microbial species. The O2 indicator is also successfully evaluated using a porcine skin wound model inoculated with E. coli. The results of this work are compared to other reported, usually invasive, smart wound monitoring systems. The possible use of this new, non-invasive smart-wound dressing technology, both at the point of care and at home, are discussed briefly.<br/

Autoimmune disease and COVID-19: a multicentre observational study in the United Kingdom

Abstract Objective To establish the demographic characteristics, laboratory findings and clinical outcomes in patients with autoimmune disease (AD) compared with a propensity-matched cohort of patients without AD admitted with COVID-19 to hospitals in the UK. Methods This is a multicentre observational study across 26 NHS Trusts. Data were collected both retrospectively and prospectively using a predesigned standardized case record form. Adult patients (≥18 years) admitted between 1 April 2020 and 31 July 2020 were included. Results Overall, 6288 patients were included to the study. Of these, 394 patients had AD prior to admission with COVID-19. Of 394 patients, 80 patients with SLE, RA or aPL syndrome were classified as severe rheumatologic AD. A higher proportion of those with AD had anaemia [240 (60.91%) vs 206 (52.28%), P = 0.015], elevated LDH [150 (38.08%) vs 43 (10.92%), P &amp;lt; 0.001] and raised creatinine [122 (30.96%) vs 86 (21.83%), P = 0.01], respectively. A significantly higher proportion of patients with severe rheumatologic AD had elevated CRP [77 (96.25%) vs 70 (87.5%), P = 0.044] and LDH [20 (25%) vs 6 (7.5%), P = 0.021]. Patients with severe rheumatologic AD had significantly higher mortality [32/80 (40%)] compared with propensity matched cohort of patients without AD [20/80 (25%), P = 0.043]. However, there was no difference in 180-day mortality between propensity-matched cohorts of patients with or without AD in general (P = 0.47). Conclusions Patients with severe rheumatologic AD had significantly higher mortality. Anaemia, renal impairment and elevated LDH were more frequent in patients with any AD while elevated CRP and LDH were more frequent in patients with severe rheumatologic AD both of which have been shown to associate with increased mortality in patients with COVID-19. </jats:sec

Author Correction: Robust estimation of bacterial cell count from optical density

An amendment to this paper has been published and can be accessed via a link at the top of the paper.</jats:p