Mucosa-Associated Bacterial Diversity in Necrotizing Enterocolitis

Background

Previous studies of infant fecal samples have failed to clarify the role of gut bacteria in the pathogenesis of NEC. We sought to characterize bacterial communities within intestinal tissue resected from infants with and without NEC.

Methods

26 intestinal samples were resected from 19 infants, including 16 NEC samples and 10 non-NEC samples. Bacterial 16S rRNA gene sequences were amplified and sequenced. Analysis allowed for taxonomic identification, and quantitative PCR was used to quantify the bacterial load within samples.

Results

NEC samples generally contained an increased total burden of bacteria. NEC and non-NEC sample sets were both marked by high inter-individual variability and an abundance of opportunistic pathogens. There was no statistically significant distinction between the composition of NEC and non-NEC microbial communities. K-means clustering enabled us to identify several stable clusters, including clusters of NEC and midgut volvulus samples enriched with Clostridium and Bacteroides. Another cluster containing both NEC and non-NEC samples was marked by an abundance of Enterobacteriaceae and decreased diversity among NEC samples.

Conclusions

The results indicate that NEC is a disease without a uniform pattern of microbial colonization, but that NEC is associated with an abundance of strict anaerobes and a decrease in community diversity.     

Temperament clusters in a normal population: implications for health and disease

Background

The object of this study was to identify temperament patterns in the Finnish population, and to determine the relationship between these profiles and life habits, socioeconomic status, and health.

Methods/Principal Findings

A cluster analysis of the Temperament and Character Inventory subscales was performed on 3,761 individuals from the Northern Finland Birth Cohort 1966 and replicated on 2,097 individuals from the Cardiovascular Risk in Young Finns study. Clusters were formed using the k-means method and their relationship with 115 variables from the areas of life habits, socioeconomic status and health was examined.

Results

Four clusters were identified for both genders. Individuals from Cluster I are characterized by high persistence, low extravagance and disorderliness. They have healthy life habits, and lowest scores in most of the measures for psychiatric disorders. Cluster II individuals are characterized by low harm avoidance and high novelty seeking. They report the best physical capacity and highest level of income, but also high rate of divorce, smoking, and alcohol consumption. Individuals from Cluster III are not characterized by any extreme characteristic. Individuals from Cluster IV are characterized by high levels of harm avoidance, low levels of exploratory excitability and attachment, and score the lowest in most measures of health and well-being.

Conclusions

This study shows that the temperament subscales do not distribute randomly but have an endogenous structure, and that these patterns have strong associations to health, life events, and well-being.     

Genetic consequences of anagenetic speciation in Acer okamotoanum (Sapindaceae) on Ullung Island, Korea

Background and Aim

Anagenesis (also known as phyletic speciation) is an important process of speciation in endemic species of oceanic islands. We investigated genetic variation in Acer okamotoanum, an anagenetically derived species endemic to Ullung Island, South Korea, to infer genetic consequences of anagenesis in comparison with other groups that have undergone cladogenesis (and adaptive radiation).

Methods

We examined genetic variation based on eight polymorphic microsatellite markers from 145 individuals of A. okamotoanum and 134 individuals of its putative progenitor A. mono. We employed standard population genetic analyses, clustering analyses, Bayesian clustering analyses in STRUCTURE and bottleneck analyses.

Key Results

Based on both the Neighbor–Joining tree and Bayesian clustering analyses, clear genetic distinctions were found between the two species. Genetic diversity in terms of allelic richness and heterozygosity shows slightly lower levels in A. okamotoanum in comparison with A. mono. Bayesian clustering analyses showed a relatively high F-value in the cluster of A. okamotoanum, suggesting a strong episode of genetic drift during colonization and speciation. There was no clear evidence of a bottleneck based on allelic frequency distribution and excess of observed heterozygotes, but the M-ratio indicated a historical bottleneck in several populations of A. okamotoanum. No geographical genetic structure within the island was found, and the genetic variation among populations of A. okamotoanum was quite low.

Conclusions

We hypothesized that genetic consequences of oceanic-endemic plants derived via anagenesis would be quite different from those derived via cladogenesis. Populations of A. okamotoanum form a cluster and are clearly differentiated from A. mono, which suggests a single origin for the anagenetically derived island endemic. No pattern of geographical differentiation of populations occurs in A. okamotoanum, which supports the concept of initial founder populations diverging through time by accumulation of mutations in a relatively uniform environment without further specific differentiation.     

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease

Introduction

Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia.

Methods

We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q₁₀ levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells.

Results

We found reduced levels of coenzyme Q₁₀, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy.

Conclusions

These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.     

Association between alcohol consumption and symptom severity and quality of life in patients with fibromyalgia

Introduction

Although alcohol consumption is a common lifestyle behavior with previous studies reporting positive effects of alcohol on chronic pain and rheumatoid arthritis, no studies to this date have examined alcohol consumption in patients with fibromyalgia. We examined the association between alcohol consumption and symptom severity and quality of life (QOL) in patients with fibromyalgia.

Methods

Data on self-reported alcohol consumption from 946 patients were analyzed. Subjects were grouped by level of alcohol consumption (number of drinks/week): none, low (≤3), moderate (>3 to 7), and heavy (>7).Univariate analyses were used to find potential confounders, and analysis of covariance was used to adjust for these confounders. Tukey HSD pairwise comparisons were used to determine differences between alcohol groups.

Results

Five hundred and forty-six subjects (58%) did not consume alcohol. Low, moderate, and heavy levels of alcohol consumption were reported for 338 (36%), 31 (3%), and 31 patients (3%), respectively. Employment status (P <0.001), education level (P = 0.009), body mass index (P = 0.002) and opioid use (P = 0.002) differed significantly among groups with drinkers having higher education, a lower BMI, and a lower frequency of unemployment and opioid use than nondrinkers. After adjusting for these differences, the measures including the number of tender points (P = 0.01), FIQ total score (P = 0.01), physical function (P <0.001), work missed (P = 0.005), job ability (P = 0.03), and pain (P = 0.001) differed across groups, as did the SF-36 subscales of physical functioning (P <0.001), pain index (P = 0.002), general health perception (P = 0.02), social functioning (P = 0.02), and the physical component summary (P <0.001). Pairwise comparison among the 4 groups showed that the moderate and low alcohol drinkers had lower severity of fibromyalgia symptoms and better physical QOL than nondrinkers.

Conclusions

Our study demonstrates that low and moderate alcohol consumption was associated with lower fibromyalgia symptoms and better QOL compared to no alcohol consumption. The reasons for these results are unclear. Since recent studies have demonstrated that γ-Aminobutyric Acid (GABA) levels are low in fibromyalgia, and alcohol is known to be a GABA-agonist, future studies should examine whether alcohol could have a salutary effect on pain and other symptoms in fibromyalgia.     

Differences in clinical features according to Boryoung and Karp genotypes of Orientia tsutsugamushi

Background

Scrub typhus is an infectious disease caused by Orientia tsutsugamushi. The differences in virulence of O. tsutsugamushi prototypes in humans are still unknown. We investigated whether there are any differences in the clinical features of the Boryoung and Karp genotypes.

Methodology/Principal Findings

Patients infected with O. tsutsugamushi, as Boryoung and Karp clusters, who had visited 6 different hospitals in southwestern Korea were prospectively compared for clinical features, complications, laboratory parameters, and treatment responses. Infected patients in the Boryoung cluster had significantly more generalized weakness, eschars, skin rashes, conjunctival injection, high albumin levels, and greater ESR and fibrinogen levels compared to the Karp cluster. The treatment response to current antibiotics was significantly slower in the Karp cluster as compared to the Boryoung cluster.

Conclusion

The frequency of occurrence of eschars and rashes may depend on the genotype of O. tsutsugamushi.     

Unsupervised analysis of classical biomedical markers: robustness and medical relevance of patient clustering using bioinformatics tools

Motivation

It has been proposed that clustering clinical markers, such as blood test results, can be used to stratify patients. However, the robustness of clusters formed with this approach to data pre-processing and clustering algorithm choices has not been evaluated, nor has clustering reproducibility. Here, we made use of the NHANES survey to compare clusters generated with various combinations of pre-processing and clustering algorithms, and tested their reproducibility in two separate samples.

Method

Values of 44 biomarkers and 19 health/life style traits were extracted from the National Health and Nutrition Examination Survey (NHANES). The 1999–2002 survey was used for training, while data from the 2003–2006 survey was tested as a validation set. Twelve combinations of pre-processing and clustering algorithms were applied to the training set. The quality of the resulting clusters was evaluated both by considering their properties and by comparative enrichment analysis. Cluster assignments were projected to the validation set (using an artificial neural network) and enrichment in health/life style traits in the resulting clusters was compared to the clusters generated from the original training set.

Results

The clusters obtained with different pre-processing and clustering combinations differed both in terms of cluster quality measures and in terms of reproducibility of enrichment with health/life style properties. Z-score normalization, for example, dramatically improved cluster quality and enrichments, as compared to unprocessed data, regardless of the clustering algorithm used. Clustering diabetes patients revealed a group of patients enriched with retinopathies. This could indicate that routine laboratory tests can be used to detect patients suffering from complications of diabetes, although other explanations for this observation should also be considered.

Conclusions

Clustering according to classical clinical biomarkers is a robust process, which may help in patient stratification. However, optimization of the pre-processing and clustering process may be still required.     

Early PTSD Symptom Trajectories: Persistence,Recovery, and Response to Treatment: Results from the Jerusalem Trauma Outreach and Prevention Study (J-TOPS)

Context

Uncovering heterogeneities in the progression of early PTSD symptoms can improve our understanding of the disorder''s pathogenesis and prophylaxis.

Objectives

To describe discrete symptom trajectories and examine their relevance for preventive interventions.

Design

Latent Growth Mixture Modeling (LGMM) of data from a randomized controlled study of early treatment. LGMM identifies latent longitudinal trajectories by exploring discrete mixture distributions underlying observable data.

Setting

Hadassah Hospital unselectively receives trauma survivors from Jerusalem and vicinity.

Participants

Adult survivors of potentially traumatic events consecutively admitted to the hospital''s emergency department (ED) were assessed ten days and one-, five-, nine- and fifteen months after ED admission. Participants with data at ten days and at least two additional assessments (n = 957) were included; 125 received cognitive behavioral therapy (CBT) between one and nine months.

Approach

We used LGMM to identify latent parameters of symptom progression and tested the effect of CBT on these parameters. CBT consisted of 12 weekly sessions of either cognitive therapy (n = 41) or prolonged exposure (PE, n = 49), starting 29.8±5.7 days after ED admission, or delayed PE (n = 35) starting at 151.8±42.4 days. CBT effectively reduced PTSD symptoms in the entire sample.

Main Outcome Measure

Latent trajectories of PTSD symptoms; effects of CBT on these trajectories.

Results

Three trajectories were identified: Rapid Remitting (rapid decrease in symptoms from 1- to 5-months; 56% of the sample), Slow Remitting (progressive decrease in symptoms over 15 months; 27%) and Non-Remitting (persistently elevated symptoms; 17%). CBT accelerated the recovery of the Slow Remitting class but did not affect the other classes.

Conclusions

The early course of PTSD symptoms is characterized by distinct and diverging response patterns that are centrally relevant to understanding the disorder and preventing its occurrence. Studies of the pathogenesis of PTSD may benefit from using clustered symptom trajectories as their dependent variables.     

Cluster analysis in severe emphysema subjects using phenotype and genotype data: an exploratory investigation

Background

Numerous studies have demonstrated associations between genetic markers and COPD, but results have been inconsistent. One reason may be heterogeneity in disease definition. Unsupervised learning approaches may assist in understanding disease heterogeneity.

Methods

We selected 31 phenotypic variables and 12 SNPs from five candidate genes in 308 subjects in the National Emphysema Treatment Trial (NETT) Genetics Ancillary Study cohort. We used factor analysis to select a subset of phenotypic variables, and then used cluster analysis to identify subtypes of severe emphysema. We examined the phenotypic and genotypic characteristics of each cluster.

Results

We identified six factors accounting for 75% of the shared variability among our initial phenotypic variables. We selected four phenotypic variables from these factors for cluster analysis: 1) post-bronchodilator FEV₁ percent predicted, 2) percent bronchodilator responsiveness, and quantitative CT measurements of 3) apical emphysema and 4) airway wall thickness. K-means cluster analysis revealed four clusters, though separation between clusters was modest: 1) emphysema predominant, 2) bronchodilator responsive, with higher FEV₁; 3) discordant, with a lower FEV₁ despite less severe emphysema and lower airway wall thickness, and 4) airway predominant. Of the genotypes examined, membership in cluster 1 (emphysema-predominant) was associated with TGFB1 SNP rs1800470.

Conclusions

Cluster analysis may identify meaningful disease subtypes and/or groups of related phenotypic variables even in a highly selected group of severe emphysema subjects, and may be useful for genetic association studies.     

Altered microbiota associated with abnormal humoral immune responses to commensal organisms in enthesitis-related arthritis

Introduction

Prior studies have established altered microbiota and immunologic reactivity to enteric commensal organisms in inflammatory bowel disease (IBD). Since intestinal inflammation is present in a subset of patients with both pediatric and adult spondyloarthritis (SpA), we hypothesized that SpA patients may also have altered microbiota and immune responsiveness to enteric organisms.

Methods

Stool and blood specimens were collected from children with enthesitis-related arthritis (ERA) and non-inflammatory controls. DNA purified from stool was subject to PCR amplification and sequencing of the variable IV region from the 16S rDNA gene. IgA and IgG Enzyme-linked Immunosorbent Assays (ELISAs) were performed on select species of bacteria in most subjects.

Results

Twenty-five children with ERA and 13 controls were included. The ERA patients had less Faecalibacterium prausnitzii (3.8% versus 10%, P = 0.008) and lachnospiraceae family (12 versus 7.0%, P = 0.020), a statistically significant increase in bifidobacterium (1.8% versus 0%, P = 0.032) and a non-statistically significant increase in Bacteroides (21% versus 11%, P = 0.150). Akkermansia muciniphila was abundant (>2%) in 7/27 ERA patients but none of the controls (P = 0.072.) Cluster analysis revealed two clusters of ERA patients: Cluster one (n = 8) was characterized by high levels of Bacteroides genus, while a second (n = 15) cluster had similar levels as the controls. Seven of 17 (41%) of the ERA subjects in Cluster 2 compared to 0/8 of the subjects in Cluster 1 had abundant Akkermansia muciniphila (P = 0.057). Serum IgA and IgG antibody levels against F. prausnitzii and B. fragilis were similar between patients and controls, whereas the two groups showed divergent responses when the fecal relative abundances of F. prausnitzii and Bacteroides were compared individually against IgA antibody levels recognizing F. prausnitzii and B. fragilis, respectively.

Conclusion

The abundance of F. prausnitzii in the stool among patients with ERA is reduced compared to controls, and Bacteroides and A. muciniphila are identified as associative agents in subsets of ERA patients. Differences in the humoral responses to these bacteria may contribute to disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0486-0) contains supplementary material, which is available to authorized users.     

A randomized,double-blind,multicenter, placebo-controlled phase III trial to evaluate the efficacy and safety of pregabalin in Japanese patients with fibromyalgia

Introduction

Fibromyalgia is a chronic disorder characterized by widespread pain and tenderness. Prior trials have demonstrated the efficacy of pregabalin for the relief of fibromyalgia symptoms, and it is approved for the treatment of fibromyalgia in the United States. However, prior to this study, there has not been a large-scale efficacy trial in patients with fibromyalgia in Japan.

Methods

This randomized, double-blind, multicenter, placebo-controlled trial was conducted at 44 centers in Japan to assess the efficacy and safety of pregabalin for the symptomatic relief of pain in fibromyalgia patients. Patients aged ≥18 years who had met the criteria for fibromyalgia were randomized to receive either pregabalin, starting at 150 mg/day and increasing to a maintenance dose of 300 or 450 mg/day, or placebo, for 15 weeks. The primary efficacy endpoint was mean pain score at final assessment. Secondary endpoints included Patient Global Impression of Change (PGIC) together with measures of sleep, physical functioning and quality of life.

Results

A total of 498 patients (89% female) were randomized to receive either pregabalin (n = 250) or placebo (n = 248). Pregabalin significantly reduced mean pain score at final assessment (difference in mean change from baseline, compared with placebo -0.44; P = 0.0046) and at every week during the study (P <0.025). Key secondary endpoints were also significantly improved with pregabalin treatment compared with placebo, including PGIC (percentage reporting symptoms "very much improved" or "much improved", 38.6% vs 26.7% with placebo; P = 0.0078); pain visual analog scale (difference in mean change from baseline, compared with placebo -6.19; P = 0.0013); Fibromyalgia Impact Questionnaire total score (-3.33; P = 0.0144); and quality of sleep score (-0.73; P <0.0001). Treatment was generally well tolerated, with somnolence and dizziness the most frequently reported adverse events.

Conclusions

This trial demonstrated that pregabalin, at doses of up to 450 mg/day, was effective for the symptomatic relief of pain in Japanese patients with fibromyalgia. Pregabalin also improved measures of sleep and functioning and was well tolerated. These data indicate that pregabalin is an effective treatment option for the relief of pain and sleep problems in Japanese patients with fibromyalgia.

Trial Registration

ClinicalTrials.gov: NCT00830167     

Biogeography and Virulence of Staphylococcus aureus

Background

Staphylococcus aureus is commonly carried asymptomatically in the human anterior nares and occasionally enters the bloodstream to cause invasive disease. Much of the global diversity of S. aureus remains uncharacterised, and is not clear how disease propensity varies between strains, and between host populations.

Methodology

We compared 147 isolates recovered from five kindergartens in Chengdu, China, with 51 isolates contemporaneously recovered from cases of pediatric infection from the main hospital serving this community. The samples were characterised by MLST, the presence/absence of PVL, and antibiotic resistance profiling.

Principal Findings

Genotype frequencies within individual kindergartens differ, but the sample recovered from cases of disease shows a general enrichment of certain MLST genotypes and PVL positive isolates. Genotypes under-represented in the disease sample tend to correspond to a single sequence cluster, and this cluster is more common in China than in other parts of the world.

Conclusions/Significance

Virulence propensity likely reflects a synergy between variation in the core genome (MLST) and accessory genome (PVL). By combining evidence form biogeography and virulence we demonstrate the existence of a “native” clade in West China which has lowered virulence, possibility due to acquired host immunity.     

Clinical Significance of Asthma Clusters by Longitudinal Analysis in Korean Asthma Cohort

Background

We have previously identified four distinct groups of asthma patients in Korean cohorts using cluster analysis: (A) smoking asthma, (B) severe obstructive asthma, (C) early-onset atopic asthma, and (D) late-onset mild asthma.

Methods and Results

A longitudinal analysis of each cluster in a Korean adult asthma cohort was performed to investigate the clinical significance of asthma clusters over 12 months.Cluster A showed relatively high asthma control test (ACT) scores but relatively low FEV₁ scores, despite a high percentage of systemic corticosteroid use. Cluster B had the lowest mean FEV₁, ACT, and the quality of life questionnaire for adult Korean asthmatics (QLQAKA) scores throughout the year, even though the percentage of systemic corticosteroid use was the highest among the four clusters. Cluster C was ranked second in terms of FEV₁, with the second lowest percentage of systemic corticosteroid use, and showed a marked improvement in subjective symptoms over time. Cluster D consistently showed the highest FEV₁, the lowest systemic corticosteroid use, and had high ACT and QLQAKA scores.

Conclusion

Our asthma clusters had clinical significance with consistency among clusters over 12 months. These distinctive phenotypes may be useful in classifying asthma in real practice.     

History and structure of the closed pedigreed population of Icelandic Sheepdogs

Background

Dog breeds lose genetic diversity because of high selection pressure. Breeding policies aim to minimize kinship and therefore maintain genetic diversity. However, policies like mean kinship and optimal contributions, might be impractical. Cluster analysis of kinship can elucidate the population structure, since this method divides the population in clusters of related individuals. Kinship-based analyses have been carried out on the entire Icelandic Sheepdog population, a sheep-herding breed.

Results

Analyses showed that despite increasing population size and deliberately transferring dogs, considerable genetic diversity has been lost. When cluster analysis was based on kinships calculated seven generation backwards, as performed in previous studies, results differ markedly from those based on calculations going back to the founder-population, and thus invalidate recommendations based on previous research. When calculated back to the founder-population, kinship-based clustering reveals the distribution of genetic diversity, similarly to strategies using mean kinship.

Conclusion

Although the base population consisted of 36 Icelandic Sheepdog founders, the current diversity is equivalent to that of only 2.2 equally contributing founders with no loss of founder alleles in descendants. The maximum attainable diversity is 4.7, unlikely achievable in a non-supervised breeding population like the Icelandic Sheepdog. Cluster analysis of kinship coefficients can provide a supporting tool to assess the distribution of available genetic diversity for captive population management.     

The Prevalence and Characteristics of Fibromyalgia in the 2012 National Health Interview Survey

Background

Most knowledge of fibromyalgia comes from the clinical setting, where healthcare-seeking behavior and selection issues influence study results. The characteristics of fibromyalgia in the general population have not been studied in detail.

Methods

We developed and tested surrogate study specific criteria for fibromyalgia in rheumatology practices using variables from the US National Health Interview Survey (NHIS) and the modification (for surveys) of the 2010 American College of Rheumatology (ACR) preliminary fibromyalgia criteria. The surrogate criteria were applied to the 2012 NHIS and identified persons who satisfied criteria from symptom data. The NHIS weighted sample of 8446 persons represents 225.7 million US adults.

Results

Fibromyalgia was identified in 1.75% (95% CI 1.42, 2.07), or 3.94 million persons. However, 73% of identified cases self-reported a physician’s diagnosis other than fibromyalgia. Identified cases had high levels of self-reported pain, non-pain symptoms, comorbidity, psychological distress, medical costs, Social Security and work disability. Caseness was associated with gender, education, ethnicity, citizenship and unhealthy behaviors. Demographics, behaviors, and comorbidity were predictive of case status. Examination of the surrogate polysymptomatic distress scale (PSD) of the 2010 ACR criteria found fibromyalgia symptoms extending through the full length of the scale.

Conclusions

Persons identified with criteria-based fibromyalgia have severe symptoms, but most (73%) have not received a clinical diagnosis of fibromyalgia. The association of fibromyalgia-like symptoms over the full length of the PSD scale with physiological as well as mental stressors suggests PSD may be a universal response variable rather than one restricted to fibromyalgia.     

Occupational risk of tuberculosis transmission in a low incidence area

Background

To investigate the occupational risk of tuberculosis (TB) infection in a low-incidence setting, data from a prospective study of patients with culture-confirmed TB conducted in Hamburg, Germany, from 1997 to 2002 were evaluated.

Methods

M. tuberculosis isolates were genotyped by IS6110 RFLP analysis. Results of contact tracing and additional patient interviews were used for further epidemiological analyses.

Results

Out of 848 cases included in the cluster analysis, 286 (33.7%) were classified into 76 clusters comprising 2 to 39 patients. In total, two patients in the non-cluster and eight patients in the cluster group were health-care workers. Logistic regression analysis confirmed work in the health-care sector as the strongest predictor for clustering (OR 17.9). However, only two of the eight transmission links among the eight clusters involving health-care workers had been detected previously. Overall, conventional contact tracing performed before genotyping had identified only 26 (25.2%) of the 103 contact persons with the disease among the clustered cases whose transmission links were epidemiologically verified.

Conclusion

Recent transmission was found to be strongly associated with health-care work in a setting with low incidence of TB. Conventional contact tracing alone was shown to be insufficient to discover recent transmission chains. The data presented also indicate the need for establishing improved TB control strategies in health-care settings.     

Statistical Cluster Analysis of the British Thoracic Society Severe Refractory Asthma Registry: Clinical Outcomes and Phenotype Stability

Background

Severe refractory asthma is a heterogeneous disease. We sought to determine statistical clusters from the British Thoracic Society Severe refractory Asthma Registry and to examine cluster-specific outcomes and stability.

Methods

Factor analysis and statistical cluster modelling was undertaken to determine the number of clusters and their membership (N = 349). Cluster-specific outcomes were assessed after a median follow-up of 3 years. A classifier was programmed to determine cluster stability and was validated in an independent cohort of new patients recruited to the registry (n = 245).

Findings

Five clusters were identified. Cluster 1 (34%) were atopic with early onset disease, cluster 2 (21%) were obese with late onset disease, cluster 3 (15%) had the least severe disease, cluster 4 (15%) were the eosinophilic with late onset disease and cluster 5 (15%) had significant fixed airflow obstruction. At follow-up, the proportion of subjects treated with oral corticosteroids increased in all groups with an increase in body mass index. Exacerbation frequency decreased significantly in clusters 1, 2 and 4 and was associated with a significant fall in the peripheral blood eosinophil count in clusters 2 and 4. Stability of cluster membership at follow-up was 52% for the whole group with stability being best in cluster 2 (71%) and worst in cluster 4 (25%). In an independent validation cohort, the classifier identified the same 5 clusters with similar patient distribution and characteristics.

Interpretation

Statistical cluster analysis can identify distinct phenotypes with specific outcomes. Cluster membership can be determined using a classifier, but when treatment is optimised, cluster stability is poor.     

Endogenous murine leukemia retroviral variation across wild European and inbred strains of house mouse

Background

Endogenous murine leukemia retroviruses (MLVs) are high copy number proviral elements difficult to comprehensively characterize using standard low throughput sequencing approaches. However, high throughput approaches generate data that is challenging to process, interpret and present.

Results

Next generation sequencing (NGS) data was generated for MLVs from two wild caught Mus musculus domesticus (from mainland France and Corsica) and for inbred laboratory mouse strains C3H, LP/J and SJL. Sequence reads were grouped using a novel sequence clustering approach as applied to retroviral sequences. A Markov cluster algorithm was employed, and the sequence reads were queried for matches to specific xenotropic (Xmv), polytropic (Pmv) and modified polytropic (Mpmv) viral reference sequences.

Conclusions

Various MLV subtypes were more widespread than expected among the mice, which may be due to the higher coverage of NGS, or to the presence of similar sequence across many different proviral loci. The results did not correlate with variation in the major MLV receptor Xpr1, which can restrict exogenous MLVs, suggesting that endogenous MLV distribution may reflect gene flow more than past resistance to infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1766-z) contains supplementary material, which is available to authorized users.     

Exploration of Chlamydial Type III Secretion System Reconstitution in Escherichia coli

Background

Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli.

Results

We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli.

Conclusions

We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity.