Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment.Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome.These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies

Blast mines: physics, injury mechanisms and vehicle protection.

Since World War II, more vehicles have been lost to land mines than all other threats combined. Anti-vehicular (AV) mines are capable of disabling a heavy vehicle, or completely destroying a lighter vehicle. The most common form of AV mine is the blast mine, which uses a large amount of explosive to directly damage the target. In a conventional military setting, landmines are used as a defensive force-multiplier and to restrict the movements of the opposing force. They are relatively cheap to purchase and easy to acquire, hence landmines are also potent weapons in the insurgents armamentarium. The stand-offnature of its design has allowed insurgents to cause significant injuries to security forces in current conflicts with little personal risk. As a result, AV mines and improvised explosive devices (IEDs) have become the most common cause of death and injury to Coalition and local security forces operating in Iraq and Afghanistan. Detonation of an AV mine causes an explosive, exothermic reaction which results in the formation of a shockwave followed by a rapid expansion of gases. The shockwave is mainly reflected by the soillair interface and fractures the soil cap overthe mine. The detonation products then vent through the voids in the soil, resulting in a hollow inverse cone which consists of the detonation gases surrounded by the soil ejecta. It is the combination of the detonation products and soil ejecta that interact with the target vehicle and cause injury to the vehicle occupants. A number of different strategies are required to mitigate the blast effects of an explosion. Primary blast effects can be reduced by increasing the standoff distance between the seat of the explosion and the crew compartment. Enhancement of armour on the base of the vehicle, as well as improvements in personal protection can prevent penetration of fragments. Mitigating tertiary effects can be achieved by altering the vehicle geometry and structure, increasing vehicle mass, as well as developing new strategies to reduce the transfer of the impulse through the vehicle to the occupants. Protection from thermal injury can be provided by incorporating fire resistant materials into the vehicle and in personal clothing. The challenge for the vehicle designer is the incorporation of these protective measures within an operationally effective platform.Published versio

Characterization of the fecal microbiota of pigs before and after inoculation with "Brachyspira hampsonii".

Brachyspira hampsonii causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with B. hampsonii , and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at -8, -5, -3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted

Comparison of the Swedish STarT Back Screening Tool and the Short Form of the Örebro Musculoskeletal Pain Screening Questionnaire in patients with acute or subacute back and neck pain

BACKGROUND: Patients with back and neck pain are often seen in primary care and it is important to provide them with tailored interventions based on risk stratification/triage. The STarT Back Screening Tool (SBT) is a widely used screening questionnaire which has not yet been validated for a population with back and/or neck pain with short duration. Our aim was to compare the concurrent validity of the SBT and the short form of the ÖMPSQ including psychometric properties and clinical utility in a primary care setting. METHODS: Patients who applied for physiotherapy by direct access (January 2013 to January 2014) at 35 primary care centers in south Sweden, with acute or subacute back and/or neck pain, aged 18–67 years, who were not currently on sick leave or had been on sick leave less than 60 days were asked to complete the SBT and ÖMPSQ-short questionnaire (n = 329). We used the Spearman’s rank correlations to study correlations, cross tabulation and Cohen’s kappa to analyze agreement of patient classification. Clinical utility was described as clinician scoring miscalculations and misclassifications of total and/or subscale scores. RESULTS: Completed SBT (9-items) and ÖMPSQ-short (10-items) data were available for 315/329 patients respectively. The statistical correlation for SBT and ÖMPSQ-short total scores was moderately strong (0.62, p < 0.01). In subgroup analyses, the correlations were 0.69 (p < 0.01) for males and 0.57 (p < 0.01) for females. The correlations were lower among older age groups, especially females over 50 years (0.21, p = 0.11). Classification to high or low risk for long-term pain and disability had moderate agreement (κ = 0.42). Observed classification agreement was 70.2%. The SBT had fewer miscalculations (13/315) than the ÖMPSQ-short (54/315). CONCLUSIONS: The correlation between the SBT and the ÖMPSQ-short scores were moderately strong for individuals with acute or subacute back and/or neck pain. SBT seemed to be clinically feasible to use in clinical practice. We therefore suggest that SBT can be used for individuals with both BP and/or NP in primary care settings but it is important to be aware of that SBT’s agreement with the ÖMPSQ-short was poor among females aged over 50 years. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT02609750 Registered: November 18, 2015

General Practitioners' and patients' perceptions towards stratified care: a theory informed investigation

Background Stratified primary care involves changing General Practitioners’ (GPs) clinical behaviour in treating patients, away from the current stepped care approach to instead identifying early treatment options that are matched to patients’ risk of persistent disabling pain. This article explores the perspectives of UK-based GPs and patients about a prognostic stratified care model being developed for patients with the five most common primary care musculoskeletal pain presentations. The focus was on views about acceptability, and anticipated barriers and facilitators to the use of stratified care in routine practice. Methods Four focus groups and six semi-structured telephone interviews were conducted with GPs (n = 23), and three focus groups with patients (n = 20). Data were analysed thematically; and identified themes examined in relation to the Theoretical Domains Framework (TDF), which facilitates comprehensive identification of behaviour change determinants. A critical approach was taken in using the TDF, examining the nuanced interrelationships between theoretical domains. Results Four key themes were identified: Acceptability of clinical decision-making guided by stratified care; impact on the therapeutic relationship; embedding a prognostic approach within a biomedical model; and practical issues in using stratified care. Whilst within each theme specific findings are reported, common across themes was the identified relationships between the theoretical domains of knowledge, skills, professional role and identity, environmental context and resources, and goals. Through analysis of these identified relationships it was found that, for GPs and patients to perceive stratified care as being acceptable, it must be seen to enhance GPs’ knowledge and skills, not undermine GPs’ and patients’ respective identities and be integrated within the environmental context of the consultation with minimal disruption. Conclusions Findings highlight the importance of taking into account the context of general practice when intervening to support GPs to make changes to their clinical behaviour. Findings will inform further stages of the research programme; specifically, the intervention format and content of support packages for GPs participating in a future randomised controlled trial (RCT). This study also contributes to the theoretical debate on how best to encourage clinical behaviour change in general practice, and the possible role of the TDF in that process

Identifying Treatment Effect Modifiers in the STarT Back Trial: A Secondary Analysis

Identification of patient characteristics influencing treatment outcomes is a top low back pain (LBP) research priority. Results from the STarT Back Trial support the effectiveness of prognostic stratified care for LBP compared to current best care, however patient characteristics associated with treatment response have not yet been explored. The purpose of this secondary analysis was to identify treatment-effect modifiers within the STarT Back Trial at 4 months follow-up (n=688). Treatment response was dichotomized using back-specific physical disability measured by the Roland-Morris Disability Questionnaire (≥7). Candidate modifiers were identified using previous literature and evaluated using logistic regression with statistical interaction terms to provide preliminary evidence of treatment-effect modification. Socioeconomic status (SES) was identified as an effect modifier for disability outcomes (OR = 1.71, P=.028). High SES patients receiving prognostic stratified care were 2.5 times less likely to have a poor outcome compared to low SES patients receiving best current care (OR = 0.40, P=.006). Education level (OR = 1.33, P=.109) and number of pain medications (OR = 0.64, P=.140) met our criteria for effect modification with weaker evidence (0.20>P≥0.05). These findings provide preliminary evidence for SES, education, and number of pain medications as treatment-effect modifiers of prognostic stratified care delivered in the STarT Back Trial

Construct and discriminant validity of STarT Back Screening Tool – Brazilian version

Background The STarT Back Screening Tool (SBST) was developed to stratify low back pain patients according to their risk of future physical disability so that prognostic subgroups can receive matched treatments in primary care. Objective To measure the construct and discriminative validity of the SBST-Brazil questionnaire. Method A hundred and fifty one patients were recruited to test the construct and discriminative validity comparing the SBST-Brazil to the Brazilian Version of the Oswestry Disability Index (ODI), Roland Morris Disability Questionnaire (RMDQ) and Fear-Avoidance Beliefs Questionnaire-Work (FABQ-W) and Physical Activity (FABQ-PA) subscales at baseline. Spearman's rank-order correlation and area under the curve (AUC) derived from receiver operating curves (ROC) for total scores and psychosocial subscale score of the SBST-Brazil were used for construct and discriminant validity analysis, respectively. Results The SBST-Brazil total and psychosocial subscale scores had good and moderate correlation with ODI (r = 0.61; r = 0.56, respectively) and good with RMDQ (r = 0.70; r = 0.64, respectively). Both scores of the SBST-Brazil total and psychosocial subscale correlated weakly and moderately with the FABQ-PA (r = 0.28; r = 0.34, respectively) and weakly with the FABQ-W (r = 0.18; r = 0.20, respectively). The discriminant validity with AUCs for the total and psychosocial subscale scores against reference standard ranged from 0.66 for kinesiophobia to 0.88 for disability. Conclusion The SBST-Brazil showed a moderate to good correlation with the disability tools, but a weak correlation with fear-avoidance beliefs. The results of discriminant validity suggest that SBST-Brazil is able to discriminate low back pain patients with disability and fear-avoidance beliefs

Does a modified STarT Back Tool predict outcome with a broader group of musculoskeletal patients than back pain? A secondary analysis of cohort data.

OBJECTIVES: The STarT Back Tool has good predictive performance for non-specific low back pain in primary care. We therefore aimed to investigate whether a modified STarT Back Tool predicted outcome with a broader group of musculoskeletal patients, and assessed the consequences of using existing risk-group cut-points across different pain regions. SETTING: Secondary analysis of prospective data from 2 cohorts: (1) outpatient musculoskeletal physiotherapy services (PhysioDirect trial n=1887) and (2) musculoskeletal primary-secondary care interface services (SAMBA study n=1082). PARTICIPANTS: Patients with back, neck, upper limb, lower limb or multisite pain with a completed modified STarT Back Tool (baseline) and 6-month physical health outcome (Short Form 36 (SF-36)). OUTCOMES: Area under the receiving operator curve (AUCs) tested discriminative abilities of the tool's baseline score for identifying poor 6-month outcome (SF-36 lower tertile Physical Component Score). Risk-group cut-points were tested using sensitivity and specificity for identifying poor outcome using (1) Youden's J statistic and (2) a clinically determined rule that specificity should not fall below 0.7 (false-positive rate <30%). RESULTS: In PhysioDirect and SAMBA, poor 6-month physical health was 18.5% and 28.2%, respectively. Modified STarT Back Tool score AUCs for predicting outcome in back pain were 0.72 and 0.79, neck 0.82 and 0.88, upper limb 0.79 and 0.86, lower limb 0.77 and 0.83, and multisite pain 0.83 and 0.82 in PhysioDirect and SAMBA, respectively. Differences between pain region AUCs were non-significant. Optimal cut-points to discriminate low-risk and medium-risk/high-risk groups depended on pain region and clinical services. CONCLUSIONS: A modified STarT Back Tool similarly predicts 6-month physical health outcome across 5 musculoskeletal pain regions. However, the use of consistent risk-group cut-points was not possible and resulted in poor sensitivity (too many with long-term disability being missed) or specificity (too many with good outcome inaccurately classified as 'at risk') for some pain regions. The draft tool is now being refined and validated within a new programme of research for a broader musculoskeletal population. TRIAL REGISTRATION NUMBER: ISRCTN55666618; Post results

Health professionals’ and coroners’ views on less invasive perinatal and paediatric autopsy: a qualitative study

OBJECTIVE: To assess health professionals’ and coroners’ attitudes towards non-minimally and minimally invasive autopsy in the perinatal and paediatric setting. METHODS: A qualitative study using semistructured interviews. Data were analysed thematically. RESULTS: Twenty-five health professionals (including perinatal/paediatric pathologists and anatomical pathology technologists, obstetricians, fetal medicine consultants and bereavement midwives, intensive care consultants and family liaison nurses, a consultant neonatologist and a paediatric radiologist) and four coroners participated. Participants viewed less invasive methods of autopsy as a positive development in prenatal and paediatric care that could increase autopsy rates. Several procedural and psychological benefits were highlighted including improved diagnostic accuracy in some circumstances, potential for faster turnaround times, parental familiarity with imaging and laparoscopic approaches, and benefits to parents and faith groups who object to invasive approaches. Concerns around the limitations of the technology such not reaching the same levels of certainty as full autopsy, unsuitability of imaging in certain circumstances, the potential for missing a diagnosis (or misdiagnosis) and de-skilling the workforce were identified. Finally, a number of implementation issues were raised including skills and training requirements for pathologists and radiologists, access to scanning equipment, required computational infrastructure, need for a multidisciplinary approach to interpret results, cost implications, equity of access and acceptance from health professionals and hospital managers. CONCLUSION: Health professionals and coroners viewed less invasive autopsy as a positive development in perinatal and paediatric care. However, to inform implementation a detailed health economic analysis and further exploration of parental views, particularly in different religious groups, are required