Building Phylogenetic Trees From Genome Sequences With kSNP4.

Performing phylogenetic analysis with genome sequences maximizes the information used to estimate phylogenies and the resolution of closely related taxa. The use of single-nucleotide polymorphisms (SNPs) permits estimating trees without genome alignments and permits the use of data sets of hundreds of microbial genomes. kSNP4 is a program that identifies SNPs without using a reference genome, estimates parsimony, maximum likelihood, and neighbor-joining trees, and is able to annotate the discovered SNPs. kSNP4 is a command-line program that does not require any additional programs or dependencies to install or use. kSNP4 does not require any programming experience or bioinformatics experience to install and use. It is suitable for use by students through senior investigators. It includes a detailed user guide that explains all of the many features of kSNP4. In this study, we provide a detailed step-by-step protocol for downloading, installing, and using kSNP4 to build phylogenetic trees from genome sequences.

Estimating microbial population data from optical density.

The spectrophotometer has been used for decades to measure the density of bacterial populations as the turbidity expressed as optical density-OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.1. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/mL. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) to show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/mL-rather than OD-is a metric that can be used to directly compare results across experiments, labs, instruments, and species.

Sociodemographic variables as predictors of adverse outcome in SARS-CoV-2 infection: an Irish hospital experience.

INTRODUCTION: Our hospital found itself at the epicentre of the Irish COVID-19 pandemic. We describe the organisational challenges faced in managing the surge and identified risk factors for mortality and ICU admission among hospitalised SARS-CoV-2-infected patients. METHODS: All hospitalised SARS-CoV-2 patients diagnosed between March 13 and May 1, 2020, were included. Demographic, referral, deprivation, ethnicity and clinical data were recorded. Multivariable regression, including age-adjusted hazard ratios (HR (95% CI), was used to explore risk factors associated with adverse outcomes. RESULTS: Of 257 inpatients, 174 were discharged (68%) and 39 died (15%) in hospital. Two hundred three (79%) patients presented from the community, 34 (13%) from care homes and 20 (8%) were existing inpatients. Forty-five percent of community patients were of a non-Irish White or Black, Asian or minority ethnic (BAME) population, including 34 Roma (13%) compared to 3% of care home and 5% of existing inpatients, (p < 0.001). Twenty-two patients were healthcare workers (9%). Of 31 patients (12%) requiring ICU admission, 18 were discharged (58%) and 7 died (23%). Being overweight/obese HR (95% CI) 3.09 (1.32, 7.23), p = 0.009; a care home resident 2.68 (1.24, 5.6), p = 0.012; socioeconomically deprived 1.05 (1.01, 1.09), p = 0.012; and older 1.04 (1.01, 1.06), p = 0.002 were significantly associated with death. Non-Irish White or BAME were not significantly associated with death 1.31 (0.28, 6.22), p = 0.63 but were significantly associated with ICU admission 4.38 (1.38, 14.2), p = 0.014 as was being overweight/obese 2.37 (1.37, 6.83), p = 0.01. CONCLUSION: The COVID-19 pandemic posed unprecedented organisational issues for our hospital resulting in the greatest surge in ICU capacity above baseline of any Irish hospital. Being overweight/obese, a care home resident, socioeconomically deprived and older were significantly associated with death, while ethnicity and being overweight/obese were significantly associated with ICU admission.

Barriers to the Recognition of Geriatric Depression in Residential Care Facilities in Alberta.

This study explored the barriers that regulated nurse professionals encountered in recognizing and assessing geriatric depression in residential care facilities in the Canadian province of Alberta. The study used a convergent parallel mixed methods design, including a cross-sectional survey (N = 635) and qualitative interviews (N = 14) with regulated nurse professionals. Findings revealed six major barriers to the recognition of geriatric depression in Alberta, including 1) insufficient clinical knowledge and training in geriatric depression; 2) misconceived beliefs about geriatric depression; 3) limited access to resources; 4) unclear depression assessment protocol and procedures in facilities; 5) characteristics of models of care and organizational culture in facilities; and 6) communication difficulties among all stakeholders in the process. Socio-cultural values and beliefs about geriatric depression played a key role in the complex interaction of the various structural and agential barriers to the effective recognition and assessment of depression in residential care facilities in Alberta.

Systematic Review and Network Meta-Analysis of Medical Therapies to Prevent Recurrence of Post-Operative Crohn's Disease.

BACKGROUND AND AIMS: Surgery is an important treatment for Crohn's disease [CD], but recurrence occurs in up to 80% of individuals post-operatively. The efficacy of several drugs to prevent post-operative recurrence has been studied in previous meta-analyses, but a number of randomized controlled trials [RCTs] have recently been published. We therefore performed an updated systematic review and network meta-analysis to investigate this issue. METHODS: We performed a comprehensive literature search through to July 2018 to identify RCTs investigating the endoscopic and clinical recurrence of CD at 12 months post-operatively. We performed a random-effects network meta-analysis to produce a pooled relative risk [RR] with 95% confidence intervals [CIs]. We ranked the treatments according to their P-score. RESULTS: We included 10 RCTs, containing 751 patients, in our primary analysis of endoscopic recurrence of CD at 12 months. Anti-tumour necrosis factor [TNF]-α therapies were significantly better than placebo, either alone [P-score 0.98, RR 0.13; 95% CI 0.04-0.39] or in combination with 5-aminosalicylates [5-ASAs] [P-score 0.81, RR 0.30; 95% CI 0.12-0.75], or 5-nitroimidazoles [P-score 0.75, RR 0.40; 95% CI 0.23-0.69]. Combination therapy with a thiopurine and 5-nitroimidazole was also more effective than placebo [P-score 0.59, RR 0.56; 95% CI 0.40-0.80], as was thiopurine monotherapy [P-score 0.31, RR 0.84; 95% CI 0.74-0.94]. However, neither 5-nitroimidazoles nor 5-ASAs alone were superior to placebo. CONCLUSIONS: In network meta-analysis, anti-TNF-α therapies alone, or in combination, appear to be the best medications for preventing endoscopic post-operative recurrence of CD.

The Effect of Antidepressants on the Course of Inflammatory Bowel Disease.

Background and Aims: Mood may have an important role in the natural history of inflammatory bowel disease (IBD). However, the impact of antidepressant use on prognosis is unknown. We aimed to address this in a longitudinal study in a referral population. Methods: We collected demographic data, clinical disease activity and mood using validated questionnaires, and antidepressant use at baseline. Longitudinal disease activity was defined by disease flare or need for glucocorticosteroids, escalation of medical therapy, hospitalisation, or intestinal resection. We compared rates of these over a minimum period of 2 years according to antidepressant use at baseline. Results: In total, 331 patients provided complete data, of whom 54 (15.8%) were taking an antidepressant at study entry. Older age, female gender, and abnormal mood scores were associated with antidepressant use. During longitudinal follow-up, there was a trend towards lower rates of any of the four endpoints of IBD activity of interest in patients with abnormal anxiety scores at baseline and who were receiving an antidepressant (42.3% versus 64.6%, P = 0.05). Based on univariate Cox regression analysis, there was a trend towards lower rates of escalation of medical therapy among patients receiving antidepressants at baseline (hazard ratio (HR) = 0.59; 95% confidence interval (CI) 0.35-1.00, P = 0.05). None of the differences observed persisted after multivariate Cox regression. Conclusions: Antidepressants may have some beneficial effects on the natural history of IBD, but larger studies with longer follow-up are required. Whether these effects are limited to patients with abnormal mood remains uncertain.

Development and validation of the Moral Distress in Dementia Care Survey instrument.

AIMS: To report on the development and validation of the Moral Distress in Dementia Care Survey instrument. BACKGROUND: Despite growing awareness of moral distress among nurses, little is known about the moral distress experiences of nursing staff in dementia care settings. To address this gap, our research team developed a tool for measuring the frequency, severity and effects of moral distress in nursing staff working in dementia care. DESIGN: The research team employed an exploratory sequential mixed method design to generate items for the moral distress questionnaire. Data were collected between January 2013 - June 2014. In this paper, we report on the development and validation of the Moral Distress in Dementia Care Survey instrument. METHODS: The Moral Distress in Dementia Care Survey instrument was piloted with a portion of the target population prior to a broader implementation. Appropriate statistical analyses and psychometric testing were completed. RESULTS: The team collected 389 completed surveys from registered nurses, licensed practical nurses and healthcare aides, representing a 43.6% response rate across 23 sites. The Moral Distress in Dementia Care Survey emerged as a reliable and valid instrument to measure the frequency, severity and effects of moral distress for nursing staff in dementia care settings. The relative value of the Moral Distress in Dementia Care Survey as a measurement instrument was superseded by its clinical relevance for dementia care staff. CONCLUSION: The Moral Distress in Dementia Care Survey is a potentially useful tool for estimating the frequency, severity and effects of moral distress in nursing staff working in dementia care settings and for the evaluation of measures taken to mitigate moral distress.

Caring in the wake of the rising tide: Moral distress in residential nursing care of people living with dementia.

Objective Moral distress is increasingly being recognized as a concern for health care professionals. The purpose of this study was to explore the nature and prevalence of moral distress among nursing staff who care for people living with dementia. Methods This study was focused on nursing staff caring for people with dementia in long-term care and assisted living sites. The Moral Distress in Dementia Care Survey instrument was distributed to 23 sites and nursing staff rated the frequency and severity of situations that were identified as potentially causing moral distress. Results Moral distress is prevalent in the nursing staff who provide dementia care. Nursing staff reported experiencing moral distress at least daily or weekly. Both frequency and severity of moral distress increased with proximity to (amount of time spent at) the bedside. Moral distress had negative psychological and physiological effects on nursing staff, and affected intention to quit.

Statistical Package for Growth Rates Made Easy.

Growth rates are an important tool in microbiology because they provide high throughput fitness measurements. The release of GrowthRates, a program that uses the output of plate reader files to automatically calculate growth rates, has facilitated experimental procedures in many areas. However, many sources of variation within replicate growth rate data exist and can decrease data reliability. We have developed a new statistical package, CompareGrowthRates (CGR), to enhance the program GrowthRates and accurately measure variation in growth rate data sets. We define a metric, Variability-score (V-score), that can help determine if variation within a data set might result in false interpretations. CGR also uses the bootstrap method to determine the fraction of bootstrap replicates in which a strain will grow the fastest. We illustrate the usage of CGR with growth rate data sets similar to those in Mira, Meza, et al. (Adaptive landscapes of resistance genes change as antibiotic concentrations change. Mol Biol Evol. 32(10): 2707-2715). These statistical methods are compatible with the analytic methods described in Growth Rates Made Easy and can be used with any set of growth rate output from GrowthRates.

One-Year Clinical Outcomes in an IBD Cohort Who Have Previously Had Anti-TNFa Trough and Antibody Levels Assessed.

BACKGROUND: Loss of response (LOR) is a big concern for anti-TNFa therapies in inflammatory bowel disease. Immunomonitoring may be useful to optimize response rates and overcome secondary LOR. METHODS: This was an observational retrospective cohort study of a group of patients with inflammatory bowel disease on infliximab (IFX) and adalimumab (ADA) who had anti-TNFa trough and antibody levels measured, during maintenance phase of treatment. Anti-TNFa trough and antibody levels were measured using standard enzyme-linked immunosorbent assay techniques. Baseline patient characteristics were determined and patients were reviewed 1 year later. Clinical assessment took place with partial Mayo scores for ulcerative colitis and Harvey-Bradshaw index for Crohn's disease. C-reactive protein (CRP) and albumin were also measured. Poor outcomes were defined as the following: need for rescue steroids, dose intensification, surgery, or treatment discontinuation. RESULTS: Seventy-four patients were included in the study, 37 (50%) were female, mean age 41 years, 61 (82%) had Crohn's disease, and 42 (57%) ulcerative colitis. Forty-two (57%) patients received IFX and 32 (43%) ADA. Mean IFX trough was 3.6 µg/mL and mean ADA troughs were 3.78 µg/mL. Twenty-seven percent of patients (n = 20) overall had a poor outcome, with a similar proportion in each group 24% (n = 10) IFX and 31% (n = 10) ADA (P value 0.24). Of the cohort, 14.2% (6/42) treated with IFX had subtherapeutic trough levels, 6.2% (2/32) of ADA patients had a trough level <1 µg/mL (P value = 0.273) There was no difference in mean trough according to outcome (4.9 µg/mL poor versus 5.4 µg/mL good, P value 0.14). Low IFX trough levels did correlate with high CRP, low albumin and response rates, mean CRP 6.66 µg/mL (n = 3), mean albumin 37 g/L for patients with low trough levels and poor response versus CRP 2.0 µg/mL (n = 24), mean albumin 43 g/L for patients with high trough levels and good response (P = 0.009, 95% confidence interval, -0.78 to -0.12). CONCLUSIONS: LOR is still a big concern with anti-TNFa therapies. Stand-alone anti-TNFa trough and antibody levels are not useful at predicting LOR/disease progression at 1 year, but low trough levels do correlate well with elevated CRP, hypoalbuminaemia, and poor response rates.

Population Dynamics of Staphylococcus aureus in Cystic Fibrosis Patients To Determine Transmission Events by Use of Whole-Genome Sequencing.

Strict infection control practices have been implemented for health care visits by cystic fibrosis (CF) patients in an attempt to prevent transmission of important pathogens. This study used whole-genome sequencing (WGS) to determine strain relatedness and assess population dynamics of Staphylococcus aureus isolates from a cohort of CF patients as assessed by strain relatedness. A total of 311 S. aureus isolates were collected from respiratory cultures of 115 CF patients during a 22-month study period. Whole-genome sequencing was performed, and using single nucleotide polymorphism (SNP) analysis, phylogenetic trees were assembled to determine relatedness between isolates. Methicillin-resistant Staphylococcus aureus (MRSA) phenotypes were predicted using PPFS2 and compared to the observed phenotype. The accumulation of SNPs in multiple isolates obtained over time from the same patient was examined to determine if a genomic molecular clock could be calculated. Pairs of isolates with ≤71 SNP differences were considered to be the "same" strain. All of the "same" strain isolates were either from the same patient or siblings pairs. There were 47 examples of patients being superinfected with an unrelated strain. The predicted MRSA phenotype was accurate in all but three isolates. Mutation rates were unable to be determined because the branching order in the phylogenetic tree was inconsistent with the order of isolation. The observation that transmissions were identified between sibling patients shows that WGS is an effective tool for determining transmission between patients. The observation that transmission only occurred between siblings suggests that Staphylococcus aureus acquisition in our CF population occurred outside the hospital environment and indicates that current infection prevention efforts appear effective.

Sources of moral distress for nursing staff providing care to residents with dementia.

The World Health Organization estimates the number of people living with dementia at approximately 35.6 million; they project a doubling of this number by 2030 and tripling by 2050. Although the majority of people living with a dementia live in the community, residential facility care by nursing care providers is a significant component of the dementia journey in most countries. Research has also shown that caring for persons with dementia can be emotionally, physically, and ethically challenging, and that turnover in nursing staff in residential care settings tends to be high. Moral distress has been explored in a variety of settings where nurses provide acute or intensive care. The concept, however, has not previously been explored in residential facility care settings, particularly as related to the care of persons with dementia. In this paper, we explore moral distress in these settings, using Nathaniel's definition of moral distress: the pain or anguish affecting the mind, body, or relationships in response to a situation in which the person is aware of a moral problem, acknowledges moral responsibility, makes a moral judgment about the correct action and yet, as a result of real or perceived constraints, cannot do what is thought to be right. We report findings from a qualitative study of moral distress in a single health region in a Canadian province. Our aim in this paper is to share findings that elucidate the sources of moral distress experienced by nursing care providers in the residential care of people living with dementia.

Long-acting somatostatin analogues provide significant beneficial effect in patients with refractory small bowel angiodysplasia: Results from a proof of concept open label mono-centre trial.

INTRODUCTION: Small bowel angiodysplasias account for over 50% of causes of small bowel bleeding and carry a worse prognosis than lesions located elsewhere in the gastrointestinal tract. Re-bleeding rates are high even after first-line endoscopic therapy and are associated with high levels of morbidity for affected patients. Small trials of long-acting somatostatin analogues have shown promising results but have not yet been assessed in patients with refractory small bowel disease. AIM: The purpose of this study was to assess the effect of long-acting somatostatin analogues in reducing re-bleeding rates and transfusion requirements, and improving haemoglobin levels in patients with refractory small bowel angiodysplasia. METHODS: Patients with refractory small bowel angiodysplasia were treated with 20 mg of long-acting octreotide for a minimum of three months. Response was assessed according to: rates of re-bleeding, haemoglobin levels, transfusion requirements, and side effects. RESULTS: A total of 24 patients were initially treated and 20 received at least three doses. Rates of complete, partial and non-response were 70%, 20% and 10% respectively. Average haemoglobin rates increased from 9.19 g/dl to 11.35 g/dl (p = 0.0027, 95% confidence interval (CI) -3.5 to -1.1) in the group overall and 70% remained transfusion-free after a mean treatment duration of 8.8 months. The rate of adverse events was higher than previously reported at 30%. CONCLUSION: Long-acting somatostatin analogues offer a therapeutic advantage in a significant proportion of patients with small bowel angiodysplasia. With careful patient selection and close observation, a long-acting somatostatin analogue should be considered in all patients with persistent anaemia attributable to refractory disease in conjunction with other standard treatments.

The natural history of small bowel angiodysplasia.

BACKGROUND: Small bowel angiodysplasias (SBA) account for 50% of obscure gastrointestinal bleeding. Lesions bleed recurrently and current treatments are relatively ineffective at reducing re-bleeding. Little is known about the natural history of SBA which is needed to guide treatment decisions and counsel patients on prognosis. AIM: The aim of this study is to describe the natural history of a cohort of patients with SBA. METHODS: Patients with SBA were identified retrospectively and clinical and outcome information were collected. Logistic regression analysis was performed to identify factors associated with re-bleeding. RESULTS: SBAs were found in 86 patients of which 54% (n = 47) were female, and the average age was 71.6 years. The majority (69%) had multiple lesions, mean of 2.76/patient, and 65% were located in the jejunum. Follow-up was available in 65% (n = 56). There was a significant increase in haemoglobin level from 10.05g/dL to 11.94g/dL, p < 0.001 after mean follow up of 31.9 (6-62) months. Re-bleeding events occurred in 80% (n = 45), with an average of 2.91/person. The mean interval between diagnosis and the first re-bleeding event was 10.7 months. Of the group overall, 70% (n = 40) required transfusions during follow up, and 67% required hospitalisation due to re-bleeding. About 50% received a directed treatment, including argon plasma coagulation, somatostatin analogues, or surgical resection. A total of 3.5% (n = 2) died as a direct consequence of bleeding from SBAs. Multiple lesions (p = 0.048) and valvular heart disease (p = 0.034) were predictive of re-bleeding. CONCLUSION: Our results show the significant impact of SBA on patients' morbidity, with high rates of re-bleeding, persistent anaemia and a mortality rate of 3.5%, despite the use of currently available medical and endoscopic therapies.

Effects of sequence diversity and recombination on the accuracy of phylogenetic trees estimated by kSNP.

kSNP v2 is a powerful tool for single nucleotide polymorphism (SNP) identification from complete microbial genomes and for estimating phylogenetic trees from the identified SNPs. kSNP can analyse finished genomes, genome assemblies, raw reads or any combination of those and does not require either genome alignment or reference genomes. This study uses sequence evolution simulations to evaluate the topological accuracy of kSNP trees and to assess the effects of diversity and recombination on that accuracy. The accuracies of kSNP trees are strongly affected by increasing diversity, with parsimony accuracy > maximum-likelihood accuracy > neighbour-joining accuracy. Accuracy is also strongly influenced by recombination; as recombination increases accuracy decreases. Reliable trees are arbitrarily defined as those that have ≥ 90% topological accuracy. It is determined that the best predictor of topological accuracy is the ratio of r/m, a measure of the effect of recombination, to FCK (the fraction of core kmers), a measure of diversity. Tools are available to allow investigators to determine both r/m and FCK, and the relationship between topological accuracy and the ratio of r/m to FCK is determined. The practical implication of this study is that kSNP is an effective tool for estimating phylogenetic trees from microbial genome sequences provided that both recombination and sequence diversity are within acceptable ranges.

PillCam COLON 2(©) as a pan-enteroscopic test in Crohn's disease.

A recent paper by Boal Carvalho et al demonstrates the potential of PillCam COLON 2(©) (PCC2) as a pan-enteric investigation in Crohn's disease (CD). Our own prospective data in patients with known CD also shows good correlation between PCC2 and small/large bowel investigations (R = 0.896, P < 0.0004/R = 0.6667, P < 0.035). Larger studies are warranted to prospectively validate the use of PCC2 in the investigation and monitoring of both small and large bowel CD.

A combined antral and corpus rapid urease testing protocol can increase diagnostic accuracy despite a low prevalence of Helicobacter pylori infection in patients undergoing routine gastroscopy.

BACKGROUND: The effects of an increased risk of sampling error and the lower prevalence of Helicobacter pylori infection on the diagnostic accuracy of standard invasive tests needs to be considered. Despite evidence of enhanced yield with additional biopsies, combined Rapid Urease Tests (RUTs) have not been widely adopted. We aimed to compare the diagnostic efficacy of a combined antral and corpus rapid urease test (RUT) to a single antral RUT in a low prevalence cohort. METHODS: Between August 2013 and April 2014 adult patients undergoing a scheduled gastroscopy were prospectively recruited. At endoscopy biopsies were taken and processed for single and combined RUTs, histology and culture using standard techniques. Infection was defined by positive culture or detection of Helicobacter like organisms on either antral or corpus samples. RESULTS: In all 123 patients were recruited. H. pylori prevalence was low at 36%, n = 44. There was a significant difference in positivity between single and combined RUTs, 20% (n = 25) versus 30% (n = 37), p = 0.0094, (95% CI 0.15-0.04). The number needed to treat (NNT) for an additional diagnosis of infection using a combined versus a single RUT is 4 (95% CI 2.2-11). The only factor associated with a reduction in RUT yield was regular proton pump inhibitor (PPI) use. Overall the sensitivity, specificity, positive and negative predictive value for any RUT test was 84%, 100%, 100% and 92% respectively. CONCLUSION: Our data suggests taking routine antral and corpus biopsies in conjunction with a combined RUT appears to optimizing H. pylori detection and overcome sampling error in a low prevalence population.