Development of a Novel Electrochemical Assay for the Rapid Detection of Pathogenic Fungi and Identification of Clinically Relevant Candida Species

A number of fungal species, particularly Candida species, are opportunistic pathogens capable of causing disease in humans. These range from relatively mild infections in healthy individuals (e.g. thrush) to life threatening systemic infections and colonisation of major organs in immunocompromised individuals. Existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. Atlas Genetics have developed a novel electrochemical assay for the detection of nucleic acid from target pathogens using PCR followed by hybridisation by labelled probes. The work describes the development of a suite of probes and optimisation of reaction conditions for the rapid detection of fungal pathogens using this assay. A suite of five species-specific probes was developed to detect the five most clinically relevant Candida species as well as a pan-fungal probe capable of detection of DNA from any fungal species. Additionally, since the assay was novel, the work investigated other aspects such as probe and primer design which led to the development of a quick bioinformatics approach for design of species-specific probes which is also described. The results demonstrated that species-specific detection of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei was possible, and that there was no cross reactivity exhibited by any probe with isolates from a test panel of other Candida species. The limit of detection of the assay was shown to be approximately one genome in 1ml of blood or 10 whole cells in 1ml of blood. The use of solid-state electrodes for detection provides an opportunity for miniaturisation of the assay into a robust, easily operated, portable system capable of rapid detection of pathogenic DNA in clinical samples either at point of care or in the microbiological laboratory.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Detection of <em>Candida albicans</em> DNA from blood samples using a novel electrochemical assay

The genus Candida contains a number of yeast species which are opportunistic pathogens and are associated with life-threatening infections in immunocompromised individuals. Provision of appropriate therapy relies on the rapid identification of the infecting species, and existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. We have previously developed a system for the rapid detection of yeast pathogens in clinical samples using PCR followed by hybridization with a suite of five species-specific, electrochemically labelled DNA probes. The limit of detection of the assay was shown to be 37 fg (∼1 genome) per reaction using extracted genomic DNA. We carried out a study to test the limit of detection of one of the probes, CA PR3, using blood samples from a healthy donor that were spiked with genomic DNA or with C. albicans cells. Our results demonstrated a limit of detection of 37 fg (ml blood)−1 (∼1 genome ml−1) using extracted DNA or 10 c.f.u. (ml blood)−1 using C. albicans cells, indicating that the assay is capable of detecting C. albicans nucleic acid at levels that are encountered in clinical samples.</jats:p

On the specifics of modelling of rotary forging processes

Rotary forging process, in spite of its various advantages, has still not reached industrial production scale owing to its complex nature. With the advent of sophisticated finite-element modelling capabilities, it is now possible to make rotary forging more predictable and optimise it for industrial production standards. However, modelling by nature involves a series of assumptions and simplifications that can help us make reasonable predictions. It is important to know the important factors that affect the results, and what compromises can be made, with a genuine understanding of what the compromises will result in. This paper reports some initial findings from our attempt towards robust modelling for the design of the rotary forging process. Herein, we have taken the simple case of rotary upsetting of cylinders using a custom-designed rotary forging machine and modelled it using commercial metal-forming software QForm

The community paediatric respiratory infection surveillance study protocol:a feasibility, prospective inception cohort study

INTRODUCTION: Paediatric respiratory tract infections (RTIs) are common reasons for primary care consultations and antibiotic prescribing. Locally relevant syndromic and microbiological surveillance information has the potential to improve the care of children with RTIs by normalising illness (parents) and reducing uncertainty (clinicians). Currently, most RTI studies are conducted at the point of healthcare service consultation, leaving the community burden, microbiology, symptom duration and proportion consulting largely unknown. This study seeks to establish the feasibility of (mainly online) participant recruitment and retention, and the acceptability/comparability of parent versus nurse-collected microbiological sampling, to inform the design of a future surveillance intervention study. Evidence regarding consultation rates and symptom duration is also sought. METHODS AND ANALYSIS: A community-based, feasibility prospective inception cohort study, recruiting children aged ≥3 months and <16 years and their parents via general practitioner surgery invitation letter, aiming to collect data on 300 incident RTIs by July 2016. Following informed consent, parents provide baseline (demographic) data online, and respond to weekly emails to confirm the absence/presence of new RTI symptoms. Once symptomatic, parents provide daily data online (RTI symptoms, school/day-care attendance, time off work, health service use, medication), and a research nurse visits to collect clinical examination data and microbiological (nasal and saliva) swabs. Parents are invited to provide symptomatic (at nurse visit, but without nurse assistance) and asymptomatic (alone) swabs on recovery. A review of primary care medical notes will gather medical history, health service utilisation, referral and antibiotic prescribing rates. Feasibility will be assessed using recruitment and retention rates, data completeness; and acceptability by quantitative survey and qualitative interviews. Symptomatic parent and nurse swab pairs will be compared for microbe isolation

Characteristics of patients with diabetic retinopathy in Gaborone, Botswana

Background: Diabetic retinopathy is a cause of preventable blindness globally and is an increasing public health problem in the developing countries. The Botswana National Screening Programme for diabetic retinopathy was launched in October 2009. We report the descriptive epidemiology of diabetic retinopathy in Botswana.Methods: The study population comprised patients on the National Diabetic Retinopathy Screening register at Princess Marina Hospital, Gaborone.  Prevalence of diabetic retinopathy, maculopathy and visual impairment were estimated.  Associations of diabetic retinopathy and explanatory variables were explored using logistic regression.Results: Of the 1,307 patients screened for diabetic retinopathy between October 2009 and August 2011, 67.9% were female and mean age (standard deviation) was 55.0 (14.1) years. The prevalence of DR and maculopathy was 17.7% (95% CI=15.6–19.9) and 14.7% (95% CI=12.7–16.7), respectively. The prevalence of low vision (presenting visual acuity [VA] ≥3/60 but &lt;6/18 in the better eye) and blindness (presenting VA of &lt;3/60 in the better eye) was 15.0% (95% CI=13.3–18.9) and 1.5% (95% CI=0.83–2.9), respectively. Increasing odds of DR were associated with increasing age (Ptrend=0.004), low vision (odds ratio [OR] =2.2; 95% CI=1.6–3.0), blindness (OR=4.6; 95%CI=2.6–8.1) and maculopathy (OR=15.2; 95% CI=10.9–21.3).Conclusion: Diabetic retinopathy is a common complication of diabetes amongst Batswana patients. Our findings are consistent with prevalence rates in other developing countries and underscore the importance of screening for diabetic retinopathy in developing nations.

Prognostic value of upper respiratory tract microbes in children presenting to primary care with respiratory infections:a prospective cohort study

BACKGROUND: The association between upper respiratory tract microbial positivity and illness prognosis in children is unclear. This impedes clinical decision-making and means the utility of upper respiratory tract microbial point-of-care tests remains unknown. We investigated for relationships between pharyngeal microbes and symptom severity in children with suspected respiratory tract infection (RTI). METHODS: Baseline characteristics and pharyngeal swabs were collected from 2,296 children presenting to 58 general practices in Bristol, UK with acute cough and suspected RTI between 2011–2013. Post-consultation, parents recorded the severity of six RTI symptoms on a 0–6 scale daily for ≤28 days. We used multivariable hurdle regression, adjusting for clinical characteristics, antibiotics and other microbes, to investigate associations between respiratory microbes and mean symptom severity on days 2–4 post-presentation. RESULTS: Overall, 1,317 (57%) children with complete baseline, microbiological and symptom data were included. Baseline characteristics were similar in included participants and those lacking microbiological data. At least one virus was detected in 869 (66%) children, and at least one bacterium in 783 (60%). Compared to children with no virus detected (mean symptom severity score 1.52), adjusted mean symptom severity was 0.26 points higher in those testing positive for at least one virus (95% CI 0.15 to 0.38, p<0.001); and was also higher in those with detected Influenza B (0.44, 0.15 to 0.72, p = 0.003); RSV (0.41, 0.20 to 0.60, p<0.001); and Influenza A (0.25, -0.01 to 0.51, p = 0.059). Children positive for Enterovirus had a lower adjusted mean symptom severity (-0.24, -0.43 to -0.05, p = 0.013). Children with detected Bordetella pertussis (0.40, 0.00 to 0.79, p = 0.049) and those with detected Moraxella catarrhalis (-0.76, -1.06 to -0.45, p<0.001) respectively had higher and lower mean symptom severity compared to children without these bacteria. CONCLUSIONS: There is a potential role for upper respiratory tract microbiological point-of-care tests in determining the prognosis of childhood RTIs

Population-based paediatric respiratory infection surveillance:a prospective inception feasibility cohort study

Abstract Background There is a need to reduce unnecessary general practitioner (GP) consultations and improve antibiotic stewardship in primary care. Respiratory tract infections (RTIs) in children are the most common reason for consulting and prescribing. Most RTI research is conducted at the point of consultation, leaving a knowledge gap regarding the population burden of RTIs. Methods Community-based, online prospective inception cohort study with nested qualitative study, to evaluate the feasibility and acceptability of collecting RTI symptom and microbiological data from children recruited prior to RTI onset. Results Parents of 10,310 children were invited. Three hundred thirty-one parents of 485 (4.7%) children responded and completed baseline data. Respondents were less socioeconomically deprived (p < 0.001) with younger (median ages 4 vs. 6 years, p < 0.001) children than non-respondents. The same parents reported 346 RTI episodes in 259 children, and 305 RTIs (in 225 children) were retained to parent-reported symptom resolution. Restricting analyses to the first RTI episode per family (to account for clustering effects), parents fully completed symptom diaries for 180 (87%) of 192 first illness episodes. Research nurses conducted home visits for 199 RTI episodes, collecting complete (symptomatic) swab sets in 195 (98%). Parents collected 194 (98% of 199 possible) symptomatic (during the nurse visit) and 282 (92% of 305 possible) asymptomatic swab sets (on symptom resolution, no nurse present). Interviews with 30 mothers and 11 children indicated study acceptability. Conclusions Invitation response rates were in the expected range. The high retention and qualitative evidence suggest that community-based paediatric syndromic and microbiological surveillance research is feasible