Clare F Aldus

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples

ABSTRACT Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli

Aims: The verotoxigenic Escherichia coli (VTEC) serotype most commonly associated with verotoxin (VT) production is O157:H7, but other serotypes have also been implicated in food‐borne illness. These serotypes exhibit much greater genetic and biochemical diversity than E. coli O157:H7, making screening for all VTEC difficult. Here we describe development and testing of novel multi‐analyte antibody‐based dipstick methods for presumptive detection of VTEC cells and VTs, including non‐O157 serotypes.

Modelling the growth of Clostridium perfringens during the cooling of bulk meat

A dynamic predictive model was developed to describe the effects of temperature, pH and NaCl concentration on the growth of Clostridium perfringens type A. The model for the specific growth rate was based on 81 growth curves generated in our laboratory or obtained from the publicly available ComBase database. Growth curves obtained during cooling were fitted with the dynamic model of Baranyi and Roberts. This made it possible to determine the parameter value reflecting the physiological state of C. perfringens after heating profiles typically applied to bulk meat. The model with the obtained parameters provided a good description of growth of C. perfringens in 24 heating/cooling curves generated specifically for this work (various non-isothermal treatments with a range of combinations of pH and NaCl concentration), and also for existing literature data. The dynamic model was implemented in Perfringens Predictor, a web-based application that can be accessed free of charge via www.combase.cc. It is anticipated that the use of this model and Perfringens Predictor will contribute to a reduction in the food poisoning incidence associated with C. perfringens.

Development and application of a new method for specific and sensitive enumeration of spores of nonproteolytic Clostridium botulinum types B, E, and F in foods and food materials.

ABSTRACT The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.

Systematic review of the health and societal effects of medication organisation devices

BackgroundSuboptimal medication adherence is a significant threat to public health and resources. Devices that organise weekly doses by time and day are commonly used to reduce unintentional non-adherence. However, there is limited evidence to support their use. This systematic review was conducted to evaluate current evidence for their efficacy, safety and costs.MethodsA pre-defined search of electronic databases from inception to January 2013 augmented with hand-searching was conducted. No limits were placed on publication date. Studies that compared organisation devices used by patients administering their own medication with standard medication packaging regardless of study design were eligible for inclusion. Studies that solely explored dispensing aspects of organisation devices were included whether or not they compared this to standard care. Screening of articles for inclusion and data extraction were completed independently by two reviewers with disagreements resolved by discussion. Outcomes were categorised into impact on health, medication adherence, healthcare utilisation, dispensing errors, supply procedures and costs. Risk of bias was also assessed.ResultsSeventeen studies met the inclusion criteria. Health outcomes were investigated in seven studies of which three reported a positive effect associated with organisation devices. Medication adherence was reported in eight studies of which three reported a positive effect. Three studies reported health care utilisation data but overall results are inconclusive. No optimal dispensing or supply procedures were identified. Economic assessment of the impact of organisation devices is lacking. All studies were subject to a high risk of bias.ConclusionsEvidence regarding the effects of medication organisation devices was limited, and the available evidence was susceptible to a high risk of bias. Organisation devices may help unintentional medication non-adherence and could improve health outcomes. There is a strong need for more studies that explore the impact of such devices on patients, and an equally pressing need for studies that explore the impacts on healthcare services.Trial registrationThis systematic review is registered with PROSPERO (Registration number CRD42011001718).

The feasibility of determining the effectiveness and cost-effectiveness of medication organisation devices compared with usual care for older people in a community setting: systematic review, stakeholder focus groups and feasibility randomised controlled trial

BACKGROUND Medication organisation devices (MODs) provide compartments for a patients medication to be organised into the days of the week and the recommended times the medication should be taken. AIM To define the optimal trial design for testing the clinical effectiveness and cost-effectiveness of MODs. DESIGN The feasibility study comprised a systematic review and focus groups to inform a randomised controlled trial (RCT) design. The resulting features were tested on a small scale, using a 2 × 2 factorial design to compare MODs with usual packaging and to compare weekly with monthly supply. The study design was then evaluated. SETTING Potential participants were identified by medical practices. PARTICIPANTS Aged over 75 years, prescribed at least three solid oral dosage form medications, unintentionally non-adherent and self-medicating. Participants were excluded if deemed by their health-care team to be unsuitable. INTERVENTIONS One of three MODs widely used in routine clinical practice supplied either weekly or monthly. OBJECTIVES To identify the most effective method of participant recruitment, to estimate the prevalence of intentional and unintentional non-adherence in an older population, to provide a point estimate of the effect size of MODs relative to usual care and to determine the feasibility and acceptability of trial participation. METHODS The systematic review included MOD studies of any design reporting medication adherence, health and social outcomes, resource utilisation or dispensing or administration errors. Focus groups with patients, carers and health-care professionals supplemented the systematic review to inform the RCT design. The resulting design was implemented and then evaluated through questionnaires and group discussions with participants and health-care professionals involved in trial delivery. RESULTS Studies on MODs are largely of poor quality. The relationship between adherence and health outcomes is unclear. Of the limited studies reporting health outcomes, some reported a positive relationship while some reported increased hospitalisations associated with MODs. The pre-trial focus groups endorsed the planned study design, but suggested a minimum recruitment age of 50-60 years. A total of 35.4% of patients completing the baseline questionnaire were excluded because they already used a MOD. Active recruitment yielded a higher consent rate, but passive recruitment was more cost-effective. The prevalence of intentional non-adherence was 24.7% [n = 71, 95% confidence interval (CI) 19.7% to 29.6%] of participants. Of the remaining 76 participants, 46.1% (95% CI 34.8% to 57.3%) were unintentionally non-adherent. There was no indication of a difference in adherence between the study arms. Participants reported a high level of satisfaction with the design. Five adverse/serious adverse events were identified in the MOD study arms and none was identified in the control arms. There was no discernible difference in health economic outcomes between the four study arms; the mean intervention cost was £20 per month greater for MOD monthly relative to usual supply monthly. CONCLUSIONS MOD provision to unintentionally non-adherent older people may cause medication-related adverse events. The primary outcome for a definitive MOD trial should be health outcomes. Such a trial should recruit patients by postal invitation and recruit younger patients. FUTURE WORK A study examining the association between MOD initiation and adverse effects is necessary and a strategy to safely introduce MODs should be explored. A definitive study testing the clinical effectiveness and cost-effectiveness of MODs is also required. STUDY REGISTRATION Current Controlled Trials ISRCTN 30626972 and UKCRN 12739. FUNDING This project was funded by National Institute for Health Research (NIHR) Health Technology Assessment Programme and will be published in full in Health Technology Assessment; Vol. 20, No. 50. See the NIHR Journals Library website for further project information.

THE PREVALENCE, CAUSES AND CONSEQUENCES OF UNDIAGNOSED DEMENTIA IN ENGLAND: A RECORD LINKAGE STUDY OF THE COGNITIVE FUNCTION AND AGEING STUDY II

dementia as having “diagnosed dementia” if the GH EMR contained a dementia diagnosis or dementia-related medication dispensing prior to the date of dementia detection in ACT (the index date) and as having “undiagnosed dementia” if none of these elements were present. Each “undiagnosed dementia” case was matched with three randomly selected ACT participants who did not develop dementia. Characteristics of “undiagnosed” and “no dementia” patients were compared using age-adjusted conditional logistic regression. Results:44% of dementia patients were undiagnosed. Undiagnosed dementia patients were more likely than no dementia patients to have dementia risk factors and comorbidities including cerebrovascular disease (22% vs. 10%, p<0.001), diabetes (20% vs. 13%, p<0.001), congestive heart failure (28% vs. 13%, p1⁄40.004), depression (25% vs. 14%, p<0.001) and traumatic brain injury (8% vs. 3%, p1⁄40.003). In addition, they were more likely to have had at least one emergency department visit (48% vs. 25%, p<0.001), hospitalization (31% vs. 21%, p1⁄40.024), or visit for an injury (14% vs. 6%, p1⁄40.006), and to “no-show” for a scheduled ambulatory visit (43% vs. 29%, p<0.001) in the previous two years. Conclusions: The EMR includes many elements that could be used to identify patients likely to have undiagnosed dementia. Additional studies are needed to develop an optimal predictive model and ultimately to investigate whether identifying and screening these patients could improve clinical outcomes and quality of life.

Detection limit of Clostridium botulinum spores in dried mushroom samples sourced from China

A survey of dried mushrooms (Lentinula edodes (Shiitake) and Auricularia auricula (Wood Ear)) sourced from China was carried out to determine the natural contamination of these mushrooms with spores of proteolytic Clostridium botulinum and non-proteolytic C. botulinum. The mushrooms were collected from supermarkets and retailers in 21 cities in China during October 2008. Spore loads of C. botulinum in mushrooms have a degree of uncertainty and variability and this study contributes valuable data for determining prevalence of spores of C. botulinum in mushrooms. An optimized detection protocol that combined selective enrichment culture with multiplex PCR was used to test for spores of proteolytic and non-proteolytic C. botulinum. Detection limits were calculated, using a maximum likelihood protocol, from mushroom samples inoculated with defined numbers of spores of proteolytic C. botulinum or non-proteolytic C. botulinum. Based on the maximum likelihood detection limit, it is estimated that dried mushroom A. auricula contained <550spores/kg of proteolytic C. botulinum, and <350spores/kg of non-proteolytic C. botulinum. Dried L. edodes contained <1500spores/kg of proteolytic C. botulinum and it was not possible to determine reliable detection limits for spores of non-proteolytic C. botulinum using the current detection protocol.