Optimizing mycobacteria molecular diagnostics: No decontamination! Human DNA depletion? Greener storage at 4 °C!

INTRODUCTION Tuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO's strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing. METHODS We begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored. RESULTS The best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at -20 °C and showed little difference between the two. DISCUSSION In summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at -20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC

The impact of routine open nonsuction drainage on fluid accumulation after thyroid surgery: a prospective randomised clinical trial.

Background: Thyroid drains following thyroid surgery are routinely used despite minimal supportive evidence. Our aim in this study is to determine the impact of routine open drainage of the thyroid bed postoperatively on ultrasound-determined fluid accumulation at 24 hours. Methods: We conducted a prospective randomised clinical trial on patients undergoing thyroid surgery. Patients were randomly assigned to a drain group (n = 49) or a no-drain group (n = 44) immediately prior to wound closure. Patients underwent a neck ultrasound on day 1 and day 2 postoperatively. After surgery, we evaluated visual analogue scale pain scores, postoperative analgesic requirements, self-reported scar satisfaction at 6 weeks and complications. Results: There was significantly less mean fluid accumulated in the drain group on both day 1, 16.4 versus 25.1 ml (P-value = 0.005), and day 2, 18.4 versus 25.7 ml (P-value = 0.026), following surgery. We found no significant differences between the groups with regard to length of stay, scar satisfaction, visual analogue scale pain score and analgesic requirements. There were four versus one wound infections in the drain versus no-drain groups. This finding was not statistically significant (P = 0.154). No life-threatening bleeds occurred in either group. Conclusions: Fluid accumulation after thyroid surgery was significantly lessened by drainage. However, this study did not show any clinical benefit associated with this finding in the non-emergent setting. Drains themselves showed a trend indicating that they may augment infection rates. The results of this study suggest that the frequency of acute life-threatening bleeds remains extremely low following abandoning drains. We advocate abandoning routine use of thyroid drains. Trial registration: ISRCTN94715414

Damned if they do, damned if they don't: negotiating the tricky context of anti-social behaviour and keeping safe in disadvantaged urban neighbourhoods

Young people's relationship with anti-social behaviour (ASB) is complicated. While their behaviours are often stereotyped as anti-social (e.g. ‘hanging about’), they also experience ASB in their neighbourhood. In this study, we explore young people's own perspectives on ASB, comparing results from ‘go-along’ interviews and focus groups conducted in disadvantaged neighbourhoods in Glasgow, Scotland. This article discusses how young people's everyday experience of ASB was contextualised by social factors such as cultural stereotyping of marginalised groups, poor social connectivity and spatial marginalisation within their neighbourhood. Furthermore, we found that these social factors were mutually reinforcing and interacted in a way that appeared to leave young people in a ‘no-win’ situation regarding their association with ASB. Participation in ASB and attempts to avoid such involvement were seen to involve negative consequences: participation could entail violence and spatial restrictions linked to territoriality, but avoidance could lead to being ostracised from their peer group. Regardless of involvement, young people felt that adults stereotyped them as anti-social. Our findings therefore provide support for policies and interventions aimed at reducing ASB (perpetrated by residents of all ages); in part by better ensuring that young people have a clear incentive for avoiding such behaviours

RandomiSed clinical trial assessing Use of an anti-inflammatoRy aGent in attenUating peri-operatiVe inflAmmatioN in non-meTastatic colon cancer – the S.U.R.G.U.V.A.N.T. trial

Background: Peri-operative inflammation has been extensively highlighted in cancer patients as detrimental. Treatment strategies to improve survival for cancer patients through targeting peri-operative inflammation have yet to be devised. Methods: We conducted a multi-centre, randomised controlled clinical trial using Taurolidine in non-metastatic colon cancer patients. Patients were randomly assigned to receive Taurolidine or a placebo. The primary endpoint for the study was the mean difference in day 1 IL-6 levels. Secondary clinical endpoints included rates of post-operative infections and tumor recurrence. Results: A total of 293 patients were screened for trial inclusion. Sixty patients were randomised. Twenty-eight patients were randomised to placebo and 32 patients to Taurolidine. IL-6 levels were equivalent on day 1 post-operatively in both groups. However, IL-6 levels were significantly attenuated over the 7 day study period in the Taurolidine group compared to placebo (p = 0.04). In addition, IL-6 levels were significantly lower at day 7 in the Taurolidine group (p = 0.04). There were 2 recurrences in the placebo group at 2 years and 1 in the Taurolidine group. The median time to recurrence was 19 months in the Placebo group and 38 months in the Taurolidine group (p = 0.27). Surgical site infection was reduced in the Taurolidine treated group (p = 0.09). Conclusion: Peri-operative use of Taurolidine significantly attenuated circulating IL-6 levels in the initial 7 day post-operative period in a safe manner. Future studies are required to establish the impact of IL-6 attenuation on survival outcomes in colon cancer. Trial registration: The trial was registered with EudraCT (year = 2008, registration number = 005570–12) and ISRCTN (year = 2008, registration number = 77,829,558)

IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist.

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, shown to play a role in the pathogenesis of intestinal diseases such as Inflammatory Bowel Disease (IBD). Given the link between IBD and colitis -associated cancer, as well as the involvement of other IL-1 family members in intestinal tumorigenesis, the aim of this work was to investigate whether IL-36 cytokines play a role in the pathogenesis of colon cancer. Whilst research to date has focused on the role of IL-36 family members in augmenting the immune response to induce tumour rejection, very little remains known about IL-36R signalling in tumour cells in this context. In this study we demonstrate that expression of IL-36 family member mRNA and protein are significantly increased in colorectal cancer tissue compared to adjacent non-tumour. In vitro assays showed stimulation of colon cancer cell lines with IL-36R agonists resulted in the activation of the pro-tumorigenic phenotypes of increased cellular migration, invasion and proliferation in both 2D and 3D models. In addition, the IL-36 cytokines induced strong expression of pro-inflammatory chemokines in both human and murine cell lines. Intraperitoneal injection of IL-36Ra significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice, and this was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra- treated group relative to untreated, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ tumour cells

Comprehensive mRNA Expression Profiling Distinguishes Tauopathies and Identifies Shared Molecular Pathways

Background: Understanding the aetiologies of neurodegenerative diseases such as Alzheimer's disease (AD), Pick's disease (PiD), Progressive Supranuclear Palsy (PSP) and Frontotemporal dementia (FTD) is often hampered by the considerable clinical and molecular overlap between these diseases and normal ageing. The development of high throughput genomic technologies such as microarrays provide a new molecular tool to gain insight in the complexity and relationships between diseases, as they provide data on the simultaneous activity of multiple genes, gene networks and cellular pathways. Methodology/Principal Findings: We have constructed genome wide expression profiles from snap frozen post-mortem tissue from the medial temporal lobe of patients with four neurodegenerative disorders (5 AD, 5 PSP, 5 PiD and 5 FTD patients) and 5 control subjects. All patients were matched for age, gender, ApoE-e and MAPT (tau) haplotype. From all groups a total of 790 probes were shown to be differently expressed when compared to control individuals. The results from these experiments were then used to investigate the correlations between clinical, pathological and molecular findings. From the 790 identified probes we extracted a gene set of 166 probes whose expression could discriminate between these disorders and normal ageing. Conclusions/Significance: From genome wide expression profiles we extracted a gene set of 166 probes whose expression could discriminate between neurological disorders and normal ageing. This gene set can be further developed into an accurate microarray-based classification test. Furthermore, from this dataset we extracted a disease specific set of genes and identified two aging related transcription factors (FOXO1A and FOXO3A) as possible drug targets related to neurodegenerative disease

Assessment of diffuse Lewy body disease by 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG PET)

BACKGROUND: Lewy body disease is, after Alzheimer's disease, the second most common cause of senile degenerative dementia with progressive cognitive deterioration, fluctuation of cognitive and motoric functions and psychotic symptoms. It is characterized histologically by the occurrence of Lewy bodies in allocortical, neocortical and subcortical structures. The aim of this study was to measure the cortical glucose metabolism using FDG PET (2-[18F]fluoro-2-deoxy-D-glucose position emission tomography) compared to normal subjects. PATIENTS AND METHODS: Five patients (5 m, mean age 75 y) with clinically suspected diffuse Lewy body disease (DLB) were studied with FDG PET. PET studies of the head were performed with a Siemens ECAT-ART PET-scanner with attenuation correction using 137-Cs point sources. RESULTS: We found the same distribution pattern of diffuse glucose hypometabolism in the entire cortical region with relative sparing of the primary sensory-motor cortex in all the patients. The few cases reported in the literature so far describe findings similar to ours. CONCLUSION: The pattern of diffuse glucose hypometabolism in the entire cortex including the occipital region seems to be a typical feature of DLB that is distinctive from dementia of Alzheimer's disease

Study protocol: EXERcise and Cognition In Sedentary adults with Early-ONset dementia (EXERCISE-ON)

<p>Abstract</p> <p>Background</p> <p>Although the development of early-onset dementia is a radical and invalidating experience for both patient and family there are hardly any non-pharmacological studies that focus on this group of patients. One type of a non-pharmacological intervention that appears to have a beneficial effect on cognition in older persons without dementia and older persons at risk for dementia is exercise. In view of their younger age early-onset dementia patients may be well able to participate in an exercise program. The main aim of the EXERCISE-ON study is to assess whether exercise slows down the progressive course of the symptoms of dementia.</p> <p>Methods/Design</p> <p>One hundred and fifty patients with early-onset dementia are recruited. After completion of the baseline measurements, participants living within a 50 kilometre radius to one of the rehabilitation centres are randomly assigned to either an <it>aerobic exercise program in a rehabilitation centre</it> or a <it>flexibility and relaxation program in a rehabilitation centre</it>. Both programs are applied three times a week during 3 months. Participants living outside the 50 kilometre radius are included in a feasibility study where participants join in a <it>daily physical activity program set at home making use of pedometers</it>. Measurements take place at baseline (entry of the study), after three months (end of the exercise program) and after six months (follow-up). Primary outcomes are cognitive functioning; psychomotor speed and executive functioning; (instrumental) activities of daily living, and quality of life. Secondary outcomes include physical, neuropsychological, and rest-activity rhythm measures.</p> <p>Discussion</p> <p>The EXERCISE-ON study is the first study to offer exercise programs to patients with early-onset dementia. We expect this study to supply evidence regarding the effects of exercise on the symptoms of early-onset dementia, influencing quality of life.</p> <p>Trial registration</p> <p>The present study is registered within The Netherlands National Trial Register (ref: NTR2124)</p

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance