Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination,accessory, and epigenetic profiles

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a serious infectious disease of humans and animals and a leading cause of community-acquired sepsis and pneumonia in endemic regions (Currie et al. 2010). Initially thought to be confined to Southeast Asia and Northern Australia, the prevalence of Bp appears to be spreading (Wiersinga et al. 2012), and Bp has been designated a biothreat select agent in the United States. Bp can persist in extreme environmental conditions and can infect several plant and animal hosts, including birds, dolphins, and humans (Wuthiekanun et al. 1995; Howard and Inglis 2003; Sprague and Neubauer 2004; Larsen et al. 2013). Treatment of clinical melioidosis is challenging because the bacterium is inherently resistant to many antibiotics, and Bp infections can persist in humans for more than a decade (Hayden et al. 2012; Wiersinga et al. 2012).The Bp genome comprises one of the largest and most complex bacterial genomes sequenced to date. Consisting of two large circular replicons (chromosomes) with a combined 7.2-Mb genome size (Holden et al. 2004), it contains a rich arsenal of genes related to virulence (e.g., Type III and Type VI secretion systems, polysaccharide biosynthesis clusters), metabolic pathways, and environmental adaptation (Wiersinga et al. 2012). Besides conserved regions, accessory genes on mobile elements and genomic islands may also contribute to phenotypic and clinical differences in microbial behavior (Currie et al. 2000; Sim et al. 2008). Analysis of the Bp genome has revealed previously unknown toxins and mechanisms of antibiotic resistance (Chantratita et al. 2011; Cruz-Migoni et al. 2011).Most large-scale studies of Bp genetic diversity to date have analyzed strains using multilocus sequence typing (MLST). These studies have suggested a high degree of genetic variability between Bp strains and related Burkholderia species (Cheng et al. 2008), and have shown that Bp strains belonging to different sequence types (STs) can often coexist in the same locale and sometimes even within the same sample (Pitt et al. 2007; Wuthiekanun et al. 2009). However, due to the limited number of genes analyzed by MLST, these studies cannot comment on the global proportion of genetic material shared between strains of different STs nor on the relative contribution of recombination, mutation, and horizontal gene transfer on intraspecies genetic diversity. Moreover, although previous studies have applied whole-genome sequencing (WGS) to study global patterns of Bp genetic heterogeneity and evolution, earlier Bp WGS reports have been confined to a limited number of isolates (10–12) derived from diverse geographical regions (Nandi et al. 2010), where geophysical barriers likely limit the propensity of the analyzed strains to exchange genetic material. To achieve a comprehensive understanding of genetic variation among closely related Bp strains, WGS analysis of much larger strain panels, ideally performed on strains isolated from a common region and belonging to the same (or closely related) ST groups, is required.In this study, we attempted to fill this important knowledge gap by performing WGS on 106 Bp strains drawn from a restricted Asian locale (Singapore and Malaysia). The WGS data, exceeding previous Bp WGS studies by 10-fold, enabled us to identify specific genomic clades of Bp, molecular features of Bp recombination at the whole-genome level, and accessory genome features contributing to recombination and horizontal gene transfer. We found a consistent pattern of genetic separation correlating with MLST, recombination haplotypes, shared accessory genes, and restriction modification (RM) systems. We provide evidence that restriction modification, beyond its role as defense against foreign DNA invasion, may have also partitioned the Bp species by restricting gene flow, resulting in the other observed correlations. Because RM systems are widely dispersed through the bacterial kingdom, it is possible that similar principles may apply to other bacterial species, implicating a potential role for epigenetic barriers as a driver of early incipient speciation.     

Psychological Comfort of Paramedics with Field Death Pronouncement: A National Asian Study to Prepare Paramedics for Field Termination of Resuscitation

Objectives: Paramedics' decision to terminate field resuscitation without a physician present may depend on personal and external factors. This study investigates factors associated with paramedic psychological comfort with termination of resuscitation (TOR) to inform future training. Methods: We administered an anonymous survey to all practicing paramedics in a large urban Asian Emergency Medical Services system where formal TOR training had not yet been conducted and field TOR was not routinely applied. The survey assessed psychological comfort using the validated Psychological Comfort Total (PCT) scale (summed score of 28 items, with higher scores representing greater comfort). We examined scores associated with four personal (prior resolution of personal loss, knowledge of survival probability, religious affiliation and experience with death pronouncements) and two external (location of patient and perceived trust of family) factors. Data were entered into Excel and analyzed by t-tests and ANOVA. Results: Response rate was 73.6% (254/345). Respondents were 30.3 years (mean, SD 7.1) with 7.2 years (mean, SD 5.54) of practice experience. Over 60% had been involved in 6 or more field death pronouncements in the prior 12 months. Higher PCT scores were associated with prior resolution of personal loss and knowledge of survival probability. Lower PCT scores were associated with patient location in a public place and perceived family lack of trust. PCT scores were not associated with paramedic religious affiliation or number of prior death pronouncements. Conclusions: Paramedic psychological comfort with field death pronouncement is associated with personal and external factors. Since paramedic comfort is important for protocol adoption, TOR education should target not only knowledge, but also public arena management, communication skills for engaging with families, and help paramedics resolve prior personal loss.     

Epicardial Coronary Spasm in Women With Angina Pectoris and Unobstructed Coronary Arteries Is Linked With a Positive Family History: An Observational Study

Purpose

Coronary spasm is frequently found in patients with angina and unobstructed coronaries. The pathophysiology is incompletely understood, although sex differences have been described. Often a positive family history (PFH) is encountered. We assessed the relationship between sex, coronary spasm, and a PFH for cardiovascular disease.

Treatment of Dural Carotid-Cavernous Fistulas via the Medial Ophthalmic Vein

Aim: To present a novel approach to treatment of dural carotid-cavernous fistulas via the medial ophthalmic vein.

Design: Retrospective case series.

Material and Methods: In this retrospective case series, we present 2 patients (3 eyes) with Type C dural CCFs, who had failed cannulation via the conventional transfemoral route and the transorbital superior ophthalmic vein approach. They subsequently underwent CCF occlusion via an anterior orbital approach through the medial ophthalmic veins, at the Department of Ophthalmology, National University Hospital Singapore. CCF occlusion was confirmed intraoperatively using angiography. Both patients were evaluated postoperatively for best-corrected visual acuity and resolution of clinical signs and symptoms.

Results: Successful occlusion of CCFs via the medial ophthalmic veins were achieved in all three orbits, with excellent visual and cosmetic outcomes postoperatively.

Conclusion: Dural CCFs may potentially lead to severe visual dysfunction and should be diagnosed and treated promptly. When all venous routes have been exhausted, the transorbital approach via the medial ophthalmic vein remains an excellent and viable alternative to access the fistula. Close cooperation between the orbital, anesthetic and radiological teams is essential in ensuring success of the operation.     

Shoulder injuries in the athlete

Shoulder injuries are common in the athletic population. Injuries can be a result of repetitive overhead use or from direct trauma. Common injury sites include the rotator cuff, glenohumeral joint, acromioclavicular joint, biceps tendon, scapulothoracic articulation, and sternoclavicular joint. The identification, physical exam, and treatment options of these conditions will be discussed.     

Is the Durie and Salmon diagnostic classification system for plasma cell dyscrasias still the best choice?

Summary There are a number of systems for diagnosing multiple myeloma, myeloma variants and monoclonal gammopathy of undetermined significance. We compared three systems, those according to Durie and Salmon, to Kyle and Greipp, and to the British Columbia Cancer Agency, using material from a populationbased registry of 847 patients with a paraproteinemia or multiple myeloma. Of these, 157 underwent both bone marrow and X-ray examinations and were subsequently included in our analysis. The differences between the systems were small, even though in only 64% of the cases the diagnosis according to all three systems was identical. The system used by the British Columbia Cancer Agency turned out to be the shortest and easiest system reviewed here. We propose a more frequent application of this system instead of the more commonly used Durie and Salmon and Kyle and Greipp criteria.     

Tumor trapping of 5-fluorouracil: in vivo 19F NMR spectroscopic pharmacokinetics in tumor-bearing humans and rabbits.

The pharmacokinetics of 5-fluorouracil (5FU) were studied in vivo in patients with discrete tumors and in rabbits bearing VX2 tumors by using 19F NMR spectroscopy. The human studies were conducted in a 1.5-T Magnetom magnetic resonance imager (Siemens), and the rabbit studies were conducted in a 4.7-T GE/Nicolet 33-cm bore magnet. Free 5FU was detected in the tumors of four of the six patients and in all VX2 tumors but not in normal rabbit tissues. No other metabolites were seen in these tumors, contrary to the extensive catabolism we had previously documented using 19F NMR spectroscopy in both human and animal livers. The tumor pool of free 5FU in those human tumors that trapped 5FU was determined to have a half-life of 0.4-2.1 hr, much longer than expected and significantly longer than the half-life of 5FU in blood (5-15 min), whereas the half-life of trapped 5FU in the VX2 tumors ranged from 1.05 to 1.22 hr. In this initial experience, patient response to chemotherapy may correlate with extent of trapping free 5FU in the human tumors. These studies document that NMR spectroscopy is clinically feasible in vivo, allows noninvasive pharmacokinetic analyses at a drug-target tissue in real time, and may produce therapeutically important information at the time of drug administration. Demonstration of the trapping of 5FU in tumors provides both a model for studying metabolic modulation in experimental tumors (in animals) and a method for testing modulation strategies clinically (in patients).     

Changes in Informed Consent Policy and Treatment Delays in Stroke Thrombolysis

ObjectivesThe efficacy of thrombolytic therapy with tissue plasminogen activator (tPA) is highly time dependent. Although clinical guidelines do not recommend written informed consent as it may cause treatment delays, local policy can supersede and require it. From 2014 to 2017, three out of five public hospitals in Singapore changed from written to verbal consent at different time points. We aimed to examine the association of hospital policy changes regarding informed consent on door-to-needle (DTN) times.Materials and MethodsUsing data from the Singapore Stroke Registry and surveys of local practice, we analyzed data of 915 acute ischemic stroke patients treated with tPA within 3 hours in all public hospitals between July 2014 to Dec 2017. Patient-level DTN times before and after policy changes were examined while adjusting for clinical characteristics, within-hospital clustering, and trends over time.ResultsPatient characteristics and stroke severity were similar before and after the policy changes. Overall, the median DTN times decreased from 68 to 53 minutes after the policy changes. After risk adjustment, changing from written to verbal informed consent was associated with a 5.6 minutes reduction (95% CI 1.1-10.0) in DTN times. After the policy changed, the percentage of patients with DTN ≤60 minutes and ≤45 minutes increased from 35.6% to 66.1% (adjusted OR 1.75; 95% CI 1.12-2.74) and 9.3% to 36.0% (adjusted OR 2.42; 95% CI 1.37-4.25), respectively.ConclusionChanging from written to verbal consent is associated with significant improvement in the timeliness of tPA administration in acute ischemic stroke.