Assessing the accuracy of quantitative molecular microbial profiling

This is the final version of the article. Available from MDPI via the DOI in this record.The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.The authors acknowledge funding from the European Metrology Research Programme joint research project “INFECT MET” (http://infectmet.lgcgroup.com) (an EMRP project, jointly funded by the EMRP participating countries within EURAMET and the European Union) and the UK National Measurement System for funding of this work and for the support of Thomas Laver by the BBSRC Industrial Case Studentship award BB/H016120/1

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides ‘absolute’ quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator

Applicability of control materials to support gene promoter characterization and expression in engineered cells using digital PCR

The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology “toolkit” of methods and materials to support the current standardization efforts

Data for improvement and clinical excellence: protocol for an audit with feedback intervention in long-term care

<p>Abstract</p> <p>Background</p> <p>There is considerable evidence about the effectiveness of audit coupled with feedback, although few audit with feedback interventions have been conducted in long-term care (LTC) settings to date. In general, the effects have been found to be modest at best, although in settings where there has been little history of audit and feedback, the effects may be greater, at least initially. The primary purpose of the Data for Improvement and Clinical Excellence (DICE) Long-Term Care project is to assess the effects of an audit with feedback intervention delivered monthly over 13 months in four LTC facilities. The research questions we addressed are:</p> <p indent="1">1. What effects do feedback reports have on processes and outcomes over time?</p> <p indent="1">2. How do different provider groups in LTC and home care respond to feedback reports based on data targeted at improving quality of care?</p> <p>Methods/design</p> <p>The research team conducting this study comprises researchers and decision makers in continuing care in the province of Alberta, Canada. The intervention consists of monthly feedback reports in nine LTC units in four facilities in Edmonton, Alberta. Data for the feedback reports comes from the Resident Assessment Instrument Minimum Data Set (RAI) version 2.0, a standardized instrument mandated for use in LTC facilities throughout Alberta. Feedback reports consist of one page, front and back, presenting both graphic and textual information. Reports are delivered to all staff working in the four LTC facilities. The primary evaluation uses a controlled interrupted time series design both adjusted and unadjusted for covariates. The concurrent process evaluation uses observation and self-report to assess uptake of the feedback reports. Following the project phase described in this protocol, a similar intervention will be conducted in home care settings in Alberta. Depending on project findings, if they are judged useful by decision makers participating in this research team, we plan dissemination and spread of the feedback report approach throughout Alberta.</p

Do self-reported intentions predict clinicians behaviour: a systematic review.

Background: Implementation research is the scientific study of methods to promote the systematic uptake of clinical research findings into routine clinical practice. Several interventions have been shown to be effective in changing health care professionals' behaviour, but heterogeneity within interventions, targeted behaviours, and study settings make generalisation difficult. Therefore, it is necessary to identify the 'active ingredients' in professional behaviour change strategies. Theories of human behaviour that feature an individual's "intention" to do something as the most immediate predictor of their behaviour have proved to be useful in non-clinical populations. As clinical practice is a form of human behaviour such theories may offer a basis for developing a scientific rationale for the choice of intervention to use in the implementation of new practice. The aim of this review was to explore the relationship between intention and behaviour in clinicians and how this compares to the intention-behaviour relationship in studies of non-clinicians. Methods: We searched: PsycINFO, MEDLINE, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials, Science/Social science citation index, Current contents (social & behavioural med/clinical med), ISI conference proceedings, and Index to Theses. The reference lists of all included papers were checked manually. Studies were eligible for inclusion if they had: examined a clinical behaviour within a clinical context, included measures of both intention and behaviour, measured behaviour after intention, and explored this relationship quantitatively. All titles and abstracts retrieved by electronic searching were screened independently by two reviewers, with disagreements resolved by discussion. Discussion: Ten studies were found that examined the relationship between intention and clinical behaviours in 1623 health professionals. The proportion of variance in behaviour explained by intention was of a similar magnitude to that found in the literature relating to non-health professionals. This was more consistently the case for studies in which intention-behaviour correspondence was good and behaviour was self-reported. Though firm conclusions are limited by a smaller literature, our findings are consistent with that of the non-health professional literature. This review, viewed in the context of the larger populations of studies, provides encouragement for the contention that there is a predictable relationship between the intentions of a health professional and their subsequent behaviour. However, there remain significant methodological challenges

Nursing home administrators’ perspectives on a study feedback report : a cross sectional survey

BackgroundThis project is part of the Translating Research in Elder Care (TREC) program of research, a multi-level and longitudinal research program being conducted in 36 nursing homes in three Canadian Prairie Provinces. The overall goal of TREC is to improve the quality of care for older persons living in nursing homes and the quality of work life for care providers. The purpose of this paper is to report on development and evaluation of facility annual reports (FARs) from facility administrators&rsquo; perspectives on the usefulness, meaningfulness, and understandability of selected data from the TREC survey. MethodsA cross sectional survey design was used in this study. The feedback reports were developed in collaboration with participating facility administrators. FARs presented results in four contextual areas: workplace culture, feedback processes, job satisfaction, and staff burnout. Six weeks after FARs were mailed to each administrator, we conducted structured telephone interviews with administrators to elicit their evaluation of the FARs. Administrators were also asked if they had taken any actions as a result of the FAR. Descriptive and inferential statistics, as well as content analysis for open-ended questions, were used to summarize findings. ResultsThirty-one facility administrators (representing thirty-two facilities) participated in the interviews. Six administrators had taken action and 18 were planning on taking action as a result of FARs. The majority found the four contextual areas addressed in FAR to be useful, meaningful, and understandable. They liked the comparisons made between data from years one and two and between their facility and other TREC study sites in their province. Twenty-two indicated that they would like to receive information on additional areas such as aggressive behaviours of residents and information sharing. Twenty-four administrators indicated that FARs contained enough information, while eight found FARs &lsquo;too short&rsquo;. Administrators who reported that the FAR contained enough information were more likely to take action within their facilities than administrators who reported that they needed more information. ConclusionsAlthough the FAR was brief, the presentation of the four contextual areas was relevant to the majority of administrators and prompted them to plan or to take action within their facility. <br /

The systematic guideline review: method, rationale, and test on chronic heart failure

Background: Evidence-based guidelines have the potential to improve healthcare. However, their de-novo-development requires substantial resources-especially for complex conditions, and adaptation may be biased by contextually influenced recommendations in source guidelines. In this paper we describe a new approach to guideline development-the systematic guideline review method (SGR), and its application in the development of an evidence-based guideline for family physicians on chronic heart failure (CHF). Methods: A systematic search for guidelines was carried out. Evidence-based guidelines on CHF management in adults in ambulatory care published in English or German between the years 2000 and 2004 were included. Guidelines on acute or right heart failure were excluded. Eligibility was assessed by two reviewers, methodological quality of selected guidelines was appraised using the AGREE instrument, and a framework of relevant clinical questions for diagnostics and treatment was derived. Data were extracted into evidence tables, systematically compared by means of a consistency analysis and synthesized in a preliminary draft. Most relevant primary sources were re-assessed to verify the cited evidence. Evidence and recommendations were summarized in a draft guideline. Results: Of 16 included guidelines five were of good quality. A total of 35 recommendations were systematically compared: 25/35 were consistent, 9/35 inconsistent, and 1/35 un-rateable (derived from a single guideline). Of the 25 consistencies, 14 were based on consensus, seven on evidence and four differed in grading. Major inconsistencies were found in 3/9 of the inconsistent recommendations. We re-evaluated the evidence for 17 recommendations (evidence-based, differing evidence levels and minor inconsistencies) - the majority was congruent. Incongruity was found where the stated evidence could not be verified in the cited primary sources, or where the evaluation in the source guidelines focused on treatment benefits and underestimated the risks. The draft guideline was completed in 8.5 man-months. The main limitation to this study was the lack of a second reviewer. Conclusion: The systematic guideline review including framework development, consistency analysis and validation is an effective, valid, and resource saving-approach to the development of evidence-based guidelines

Which factors explain variation in intention to disclose a diagnosis of dementia? A theory-based survey of mental health professionals

Background: For people with dementia, patient-centred care should involve timely explanation of the diagnosis and its implications. However, this is not routine. Theoretical models of behaviour change offer a generalisable framework for understanding professional practice and identifying modifiable factors to target with an intervention. Theoretical models and empirical work indicate that behavioural intention represents a modifiable predictor of actual professional behaviour. We identified factors that predict the intentions of members of older people's mental health teams (MHTs) to perform key behaviours involved in the disclosure of dementia. Design: Postal questionnaire survey. Participants: Professionals from MHTs in the English National Health Service. Methods: We selected three behaviours: Determining what patients already know or suspect about their diagnosis; using explicit terminology when talking to patients; and exploring what the diagnosis means to patients. The questionnaire was based upon the Theory of Planned Behaviour (TPB), Social Cognitive Theory (SCT), and exploratory team variables. Main outcomes: Behavioural intentions. Results: Out of 1,269 professionals working in 85 MHTs, 399 (31.4%) returned completed questionnaires. Overall, the TPB best explained behavioural intention. For determining what patients already know, the TPB variables of subjective norm, perceived behavioural control and attitude explained 29.4% of the variance in intention. For the use of explicit terminology, the same variables explained 53.7% of intention. For exploring what the diagnosis means to patients, subjective norm and perceived behavioural control explained 48.6% of intention. Conclusion: These psychological models can explain up to half of the variation in intention to perform key disclosure behaviours. This provides an empirically- supported, theoretical basis for the design of interventions to improve disclosure practice by targeting relevant predictive factors. Trial Registration: ISRCTN15871014

Angiotensin converting enzyme gene polymorphism is associated with severity of coronary artery disease in men with high total cholesterol levels

This study examines whether renin-angiotensin-aldosterone system gene polymorphisms: ACE (encoding for angiotensin converting enzyme) c.2306-117_404 I/D, AGTR1 (encoding for angiotensin II type-1 receptor) c.1080*86A>C and CYP11B2 (encoding for aldosterone synthase) c.-344C>T are associated with the extension of coronary atherosclerosis in a group of 647 patients who underwent elective coronary angiography. The extension of CAD was evaluated using the Gensini score. The polymorphisms were determined by PCR and RFLP assays. The associations between genotypes and the extent of coronary atherosclerosis were tested by the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon test. The population has been divided into groups defined by: sex, smoking habit, past myocardial infarction, BMI (>, ≤ 25), age (>, ≤ 55), diabetes mellitus, level of total cholesterol (>, ≤ 200 mg/dl), LDL cholesterol (>, ≤ 130 mg/dl), HDL cholesterol (>, ≤ 40 mg/dl), triglycerides (>, ≤ 150 mg/dl). Significant associations between the ACE c.2306-117_404 I/D polymorphism and the Gensini score in men with high total cholesterol levels (PKruskal-Wallis = 0.008; Padjusted = 0.009), high level of LDL cholesterol (PKruskal-Wallis = 0.016; Padjusted = 0.028) and low level of HDL cholesterol (PKruskal-Wallis = 0.04; Padjusted = 0.055) have been found. No association between the AGTR1 c.1080*86A>C and CYP11B2 c.-344C>T and the Gensini score has been found. These results suggest that men who carry ACE c.2306-117_404 DD genotype and have high total cholesterol, high LDL cholesterol and low HDL cholesterol levels may be predisposed to the development of more severe CAD

Laxative activities of Mareya micrantha (Benth.) Müll. Arg. (Euphorbiaceae) leaf aqueous extract in rats

<p>Abstract</p> <p>Background</p> <p><it>Mareya micrantha </it>(Benth.) Müll. Arg. (Euphorbiaceae) is a shrub that is commonly used in Côte d'Ivoire (West Africa) for the treatment of constipation and as an ocytocic drug. The present study was carried out to investigate the laxative activity of <it>Mareya micrantha </it>in albino's Wistar rats.</p> <p>Methods</p> <p>Rats were divided in 5 groups of 5 animals each, first group as control, second group served as standard (sodium picosulfate) while group 3, 4 and 5 were treated with leaf aqueous extract of <it>Mareya micrantha </it>at doses of 100, 200 and 400 mg/kg body weight (b.w.), <it>per os </it>respectively. The laxative activity was determined based on the weight of the faeces matter. The effects of the leaves aqueous extract of <it>Mareya micrantha </it>and castor oil were also evaluated on intestinal transit, intestinal fluid accumulation and ions secretion.</p> <p>Results</p> <p>Phytochemicals screening of the extract revealed the presence of flavonoids, alkaloids, tannins, polyphenols, sterols and polyterpenes. The aqueous extract of <it>Mareya micrantha </it>applied orally (100, 200 and 400 mg/kg; <it>p.o</it>.), produced significant laxative activity and reduced loperamide induced constipation in dose dependant manner. The effect of the extract at 200 and 400 mg/kg (<it>p.o</it>.) was similar to that of reference drug sodium picosulfate (5 mg/kg, <it>p.o</it>). The same doses of the extract (200 and 400 mg/kg, <it>p.o</it>.) produced a significant increase (p < 0.01) of intestinal transit in comparison with castor oil (2 mL) (p < 0.01). Moreover, the extract induced a significant enteropooling and excretion of Cl^-, Na⁺, K⁺and Ca²⁺in the intestinal fluid (p < 0.01).</p> <p>Conclusions</p> <p>The results showed that the aqueous extract of <it>Mareya micrantha </it>has a significant laxative activity and supports its traditional use in herbal medicine.</p