Management of Severe Anemia in a Jehovah's Witness Patient With Lung Abscess Secondary to Malpositioned Laparoscopic Adjustable Gastric Band: A Case Report.

Jehovah's Witnesses is a Christian denomination widely recognized for their steadfast refusal of blood transfusions, even when facing severe anemia. We describe a unique case of a 42-year-old Jehovah's Witness woman with severe iron deficiency anemia. She necessitated surgical correction of a malpositioned gastric band within the context of a complex necrotizing aspiration pneumonia secondary to esophageal obstruction. Medical management of this severe anemia has been a challenge as traditional approaches, like a blood transfusion, are not possible. Instead, a multifaceted approach has been described with intravenous iron infusions, recombinant human erythropoietin, vitamin B12, folate, and vitamin C administration. We emphasize the lack of consensus on guideline protocols regarding management of severe anemia for Jehovah's Witness patients and the subsequent need for more investigation into that matter. It also underscores the significance of respecting patient autonomy through close collaboration between patients and their health care providers to ensure effective patient-centered care.

Post Dural Puncture Subdural Hematoma: A Rare Iatrogenic Complication of Neuraxial Anesthesia.

Although post-dural puncture headache is a well-known complication of neuraxial anesthesia, subdural hematoma following epidural injection is a rare but potentially under-recognized complication. The following is the case of a young female patient with no significant past medical history who developed a persistent and severe headache following the introduction of epidural anesthesia for labor induction. Imaging demonstrated multiple small subdural hematomas with findings concerning intracranial hypotension. She eventually underwent a blood patch and reported immediate relief from her headache.

Influence of medical trainee sleep pattern (chronotype) on burn-out and satisfaction with work schedules: a multicentre observational study.

BACKGROUND: Medical trainees' work schedule is designed to cover duties without consideration of differences in circadian rhythms during a 24-hour period (chronotype). OBJECTIVE: To explore chronotype variation among medical trainees and understand its association with burn-out and schedule satisfaction. METHODS: In a multicentre observational study, we conducted two surveys between 1 October 2018 and 1 April 2019. Trainees from nine centres across the USA participated. We measured burn-out using Maslach Burnout Inventory (MBI), and trainee chronotype using the Morningness-Eveningness Questionnaire (MEQ). RESULTS: 324 (32%) out of 1012 responded to our survey. Participants were 51% female and had a mean age of 30.8 years. Most participants had an intermediate MEQ type (65%). A large proportion of participants had burn-out on at least one of three tested MBI scales (62%); 5% of participants had burn-out on all three MBI scales. More participants with evening MEQ type had burn-out (66%) compared with morning MEQ type (55%), however, the results were not statically significant (p=0.294). Overall satisfaction with work shifts was 6.5 (95% CI 6.3 to 6.7), with higher satisfaction with day shift 7.7 (95% CI 7.5 to 7.9) and lowest satisfaction with overnight 24-hour call 3.5 (95% CI 3.2 to 3.9). Satisfaction was lower in trainees with burn-out 6.0 (95% CI 5.7 to 6.4), (p<0.001). In the follow-up survey, burn-out was present in at least one scale in 64% compared with 60% of respondents in the initial survey. CONCLUSION: Burn-out is prevalent among medical trainees. Improving alignment between trainee preferences may improve performance, reduce human errors and burn-out.

Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis.

BACKGROUND: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. RESULTS: We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. CONCLUSIONS: The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

Systematic review and meta-analysis of outcomes in patients with suspected pulmonary embolism.

Prompt evaluation and therapeutic intervention of suspected pulmonary embolism (PE) are of paramount importance for improvement in outcomes. We systematically reviewed outcomes in patients with suspected PE, including mortality, incidence of recurrent PE, major bleeding, intracranial hemorrhage, and postthrombotic sequelae. We searched the Cochrane Central Register of Controlled Trials, MEDLINE, and Embase for eligible studies, reference lists of relevant reviews, registered trials, and relevant conference proceedings. We included 22 studies with 15 865 patients. Among patients who were diagnosed with PE and discharged with anticoagulation, 3-month follow-up revealed that all-cause mortality was 5.69% (91/1599; 95% confidence interval [CI], 4.56-6.83), mortality from PE was 1.19% (19/1597; 95% CI, 0.66-1.72), recurrent venous thromboembolism (VTE) occurred in 1.38% (22/1597; 95% CI: 0.81-1.95), and major bleeding occurred in 0.90% (2/221%; 95% CI, 0-2.15). In patients with a low pretest probability (PTP) and negative D-dimer, 3-month follow-up revealed mortality from PE was 0% (0/808) and incidence of VTE was 0.37% (4/1094; 95% CI: 0.007-0.72). In patients with intermediate PTP and negative D-dimer, 3-month follow-up revealed that mortality from PE was 0% (0/2747) and incidence of VTE was 0.46% (14/3015; 95% CI: 0.22-0.71). In patients with high PTP and negative computed tomography (CT) scan, 3-month follow-up revealed mortality from PE was 0% (0/651) and incidence of VTE was 0.84% (11/1302; 95% CI: 0.35-1.34). We further summarize outcomes evaluated by various diagnostic tests and diagnostic pathways (ie, D-dimer followed by CT scan).

The airways microbiome of individuals with asthma treated with high and low doses of inhaled corticosteroids.

BACKGROUND: Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period. METHODS: We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks. RESULTS: A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period. DISCUSSION: Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02671773.

Systematic review and meta-analysis of test accuracy for the diagnosis of suspected pulmonary embolism.

Pulmonary embolism (PE) is a common, potentially life-threatening yet treatable condition. Prompt diagnosis and expeditious therapeutic intervention is of paramount importance for optimal patient management. Our objective was to systematically review the accuracy of D-dimer assay, compression ultrasonography (CUS), computed tomography pulmonary angiography (CTPA), and ventilation-perfusion (V/Q) scanning for the diagnosis of suspected first and recurrent PE. We searched Cochrane Central, MEDLINE, and EMBASE for eligible studies, reference lists of relevant reviews, registered trials, and relevant conference proceedings. 2 investigators screened and abstracted data. Risk of bias was assessed using Quality Assessment of Diagnostic Accuracy Studies-2 and certainty of evidence using the Grading of Recommendations Assessment, Development and Evaluation framework. We pooled estimates of sensitivity and specificity. The review included 61 studies. The pooled estimates for D-dimer sensitivity and specificity were 0.97 (95% confidence interval [CI], 0.96-0.98) and 0.41 (95% CI, 0.36-0.46) respectively, whereas CTPA sensitivity and specificity were 0.94 (95% CI, 0.89-0.97) and 0.98 (95% CI, 0.97-0.99), respectively, and CUS sensitivity and specificity were 0.49 (95% CI, 0.31-0.66) and 0.96 (95% CI, 0.95-0.98), respectively. Three variations of pooled estimates for sensitivity and specificity of V/Q scan were carried out, based on interpretation of test results. D-dimer had the highest sensitivity when compared with imaging. CTPA and V/Q scans (high probability scan as a positive and low/non-diagnostic/normal scan as negative) both had the highest specificity. This systematic review was registered on PROSPERO as CRD42018084669.

QIPS CURE: Implementing a Quality Improvement and Patient Safety Curriculum and Resident Experience.

BACKGROUND: Resident participation in quality improvement and patient safety (QIPS) programs is an essential training experience and Accreditation Council for Graduate Medical Education requirement. However, the most effective approach to achieve this is unclear. OBJECTIVE: We developed an experiential Quality Improvement and Patient Safety Curriculum and Resident Experience (QIPS CURE) program, which provides internal medicine (IM) residents with foundational QIPS knowledge, and evaluated its effectiveness. METHODS: After reviewing IM residency QIPS curricula and obtaining input from institutional stakeholders in 2013-2014, we launched a longitudinal QIPS curriculum for all 66 postgraduate year 1-3 IM residents in July 2014. The QIPS CURE included 2 major elements: didactics, delivered through a variety of sources, including online modules and workshops, and hands-on projects. We delivered this curriculum annually from 2014 to 2018. We used project completion and an attitude survey of participants to evaluate it. RESULTS: Six projects were completed in 2014-2015, and 10 projects completed yearly for the next 3 academic years. Residents presented all projects at regional meetings. Surveyed residents reported improvement in understanding (M = 5.71, SD = 1.07 pre- to M = 6.38, SD = 0.49 post-curriculum, P = .013) and competence (M = 3.31, SD = 1.18 pre- to M = 6.08, SD = 0.77, post-curriculum, P < .001) when comparing graduates of the curriculum with incoming interns. Qualitative analysis revealed perceived acquisition of skills needed to carry out successful QIPS projects. CONCLUSIONS: This QIPS program was sustainable over 4 years and generally well-received by residents, with many projects completed each year.

Diagnosis of deep vein thrombosis of the upper extremity: a systematic review and meta-analysis of test accuracy.

Upper extremity deep vein thrombosis (UEDVT) accounts for ≤10% of DVT and can be associated with morbidity and mortality. Accurate diagnosis and treatment are necessary for safe and effective patient management. We systematically reviewed the accuracy of D-dimer and duplex ultrasonography (US) for the evaluation of suspected first-episode UEDVT. We searched the Cochrane Central Register, OVID MEDLINE, EMBASE, and PubMed for eligible studies, reference lists of relevant reviews, registered trials, and relevant conference proceedings. We included prospective cross-sectional and cohort studies that evaluated test accuracy. Two investigators independently screened and collected data. The risk of bias was assessed using Quality Assessment of Diagnostic Accuracy Studies 2 and certainty of evidence using the Grading of Recommendations Assessment, Development and Evaluation framework. We pooled estimates of sensitivity and specificity. The review included 9 studies. The pooled estimates for D-dimer sensitivity and specificity were 0.96 (95% confidence interval [CI], 0.87-0.99) and 0.47 (95% CI, 0.43-0.52), respectively. The pooled estimates for duplex US sensitivity and specificity were 0.87 (95% CI, 0.73-0.94) and 0.85 (95% CI, 0.72-0.93), respectively. Certainty of evidence was moderate. In this review, we summarized the test accuracy (sensitivity and specificity) of D-dimer and duplex US for this indication. The sensitivity and specificity of the tests found in the present review should be considered in the context of whether they are used alone or in combination, which is dependent on the prevalence of disease in the population, the clinical setting in which the patient is being evaluated, cost, potential harms, and patient outcomes. This study was registered at PROSPERO as Systematic Review Registration Number CRD42018098488.

Establishing a Multi-Institutional Quality and Patient Safety Consortium: Collaboration Across Affiliates in a Community-Based Medical School.

The landscape of health care delivery and medical education is evolving. Institutions must continually reassess priorities, strategies, and partnerships to align the knowledge and skills of the health care workforce with the delivery of quality, socially accountable, collaborative health care that meets the needs of diverse populations in communities. This article describes the development, implementation, and early outcomes of the University of Missouri-Kansas City's Health Care Quality and Patient Safety Consortium. Inspired by an actual patient safety event, the consortium aimed to improve patient outcomes by establishing quality improvement and patient safety (QIPS) education and scholarship as foundational within its unique, horizontal-matrix academic health center, which comprises 6 affiliated hospitals and 4 university-based health sciences schools. The consortium established a governance structure with leaders who, collectively, represent the diverse members and stakeholders of the consortium. The members share a common agenda and mutual goals. The consortium measures success by applying published conceptual frameworks for evaluating the outcomes of educational programs on learners (Kirkpatrick) and patients (Bzowyckyj and colleagues). Consortium learner and patient outcomes span all levels of these frameworks. Undergraduate and graduate QIPS-based projects with meaningful health system or improved individual health outcomes signify a Level 4 outcome (the highest level) for learners and patients alike. Factors critical to success include a financial gift, leadership buy-in and support, a clear champion, shared goals and a united vision, a willingness to collaborate across health systems with varied strengths and priorities, and a stable communication platform. Aspirational goals of the consortium include increasing involvement across health professional schools, incorporating simulation into QIPS activities, and aligning the consortium's projects with broader community needs.

Systematic review and meta-analysis of outcomes in patients with suspected deep vein thrombosis.

After deep vein thrombosis (DVT) is diagnosed, prompt evaluation and therapeutic intervention are of paramount importance for improvement in patient-important outcomes. We systematically reviewed patient-important outcomes in patients with suspected DVT, including mortality, incidence of pulmonary embolism (PE) and DVT, major bleeding, intracranial hemorrhage, and postthrombotic sequelae. We searched the Cochrane Central Register of Controlled Trials, Ovid Medline, Embase for eligible studies, references lists of relevant reviews, registered trials, and relevant conference proceedings. Two investigators screened and abstracted data. Nine studies with 5126 patients were included for lower extremity DVT. Three studies with 500 patients were included for upper extremity DVT. Among patients with lower extremity DVT, 0.85% (95% confidence interval [CI], 0% to 2.10%) and 0% developed recurrent DVT and PE, respectively, at 3 months. Among patients with upper extremity DVT, 0.49% (95% CI, 0% to 1.16%) and 1.98% (95% CI, 0.62% to 3.33%) developed recurrent DVT and PE, respectively, at 3 months. No major bleeding events were reported for those anticoagulated, which is lower than in other systematic reviews. For both upper and lower extremity DVT, low pretest probability patients with a negative D-dimer had a comparable incidence of VTE at 3 months (∼1%) as patients with a negative ultrasound (US). At higher pretest probabilities, negative US testing with or without serial US appears to be the safer option. In this review, we summarized the outcomes of patients evaluated by various diagnostic pathways. In most instances, there was significant limitation due to small population size or lack of direct evidence of effects of using a specific pathway. This systematic review was registered at PROSPERO as CRD42018100502.

Mobile App to Improve House Officers' Adherence to Advanced Cardiac Life Support Guidelines: Quality Improvement Study.

BACKGROUND: Effective and timely delivery of cardiac arrest interventions during in-hospital cardiac arrest resuscitation is associated with greater survival. Whether a mobile app that provides timely reminders of critical interventions improves adherence to Advanced Cardiovascular Life Support (ACLS) guidelines among house officers, who may lack experience in leading resuscitations, remains unknown. OBJECTIVE: The aim of this study was to assess the impact of a commercially available, dynamic mobile app on house officers' adherence to ACLS guidelines. METHODS: As part of a quality improvement initiative, internal medicine house officers were invited to participate and randomized to lead 2 consecutive cardiac arrest simulations, one with a novel mobile app and one without a novel mobile app. All simulations included 4 cycles of cardiopulmonary resuscitation with different cardiac arrest rhythms and were video recorded. The coprimary end points were chest compression fraction and number of correct interventions in each simulation. The secondary end point was incorrect interventions, defined as interventions not indicated by the 2015 ACLS guidelines. Paired t tests compared performance with and without the mobile app. RESULTS: Among 53 house officers, 26 house officers were randomized to lead the first simulation with the mobile app, and 27 house officers were randomized to do so without the app. Use of the mobile app was associated with a higher number of correct ACLS interventions (out of 7; mean 6.2 vs 5.1; absolute difference 1.1 [95% CI 0.6 to 1.6]; P<.001) as well as fewer incorrect ACLS interventions (mean 0.3 vs 1.0; absolute difference -0.7 [95% CI -0.3 to -1.0]; P<.001). Simulations with the mobile app also had a marginally higher chest compression fraction (mean 90.9% vs 89.0%; absolute difference 1.9% [95% CI 0.6% to 3.4%]; P=.007). CONCLUSIONS: This proof-of-concept study suggests that this novel mobile app may improve adherence to ACLS protocols, but its effectiveness on survival in real-world resuscitations remains unknown.

Diagnosis of deep vein thrombosis of the lower extremity: a systematic review and meta-analysis of test accuracy.

Deep vein thrombosis (DVT) of the lower extremities can be associated with significant morbidity and may progress to pulmonary embolism and postthrombotic syndrome. Early diagnosis and treatment are important to minimize the risk of these complications. We systematically reviewed the accuracy of diagnostic tests for first-episode and recurrent DVT of the lower extremities, including proximal compression ultrasonography (US), whole leg US, serial US, and high-sensitivity quantitative D-dimer assays. We searched Cochrane Central, MEDLINE, and EMBASE for eligible studies, reference lists of relevant reviews, registered trials, and relevant conference proceedings. Two investigators screened and abstracted data. Risk of bias was assessed using Quality Assessment of Diagnostic Accuracy Studies-2 and certainty of evidence using the Grading of Recommendations Assessment, Development and Evaluation framework. We pooled estimates of sensitivity and specificity. The review included 43 studies. For any suspected DVT, the pooled estimates for sensitivity and specificity of proximal compression US were 90.1% (95% confidence interval [CI], 86.5-92.8) and 98.5% (95% CI, 97.6-99.1), respectively. For whole-leg US, pooled estimates were 94.0% (95% CI, 91.3-95.9) and 97.3% (95% CI, 94.8-98.6); for serial US pooled estimates were 97.9% (95% CI, 96.0-98.9) and 99.8% (95% CI, 99.3-99.9). For D-dimer, pooled estimates were 96.1% (95% CI, 92.6-98.0) and 35.7% (95% CI, 29.5-42.4). Recurrent DVT studies were not pooled. Certainty of evidence varied from low to high. This systematic review of current diagnostic tests for DVT of the lower extremities provides accuracy estimates. The tests are evaluated when performed in a stand-alone fashion, and in a diagnostic pathway. The pretest probability of DVT often assessed by a clinical decision rule will influence how, together with sensitivity and specificity estimates, patients will be managed.

Improving Resident Feedback on Diagnostic Reasoning after Handovers: The LOOP Project.

Appropriate calibration of clinical reasoning is critical to becoming a competent physician. Lack of follow-up after transitions of care can present a barrier to calibration. This study aimed to implement structured feedback about clinical reasoning for residents performing overnight admissions, measure the frequency of diagnostic changes, and determine how feedback impacts learners' self-efficacy. Trainees shared feedback via a structured form within their electronic health record's secure messaging system. Forms were analyzed for diagnostic changes. Surveys evaluated comfort with sharing feedback, self-efficacy in identifying and mitigating cognitive biases' negative effects, and perceived educational value of night admissions-all of which improved after implementation. Analysis of 544 forms revealed a 43.7% diagnostic change rate spanning the transition from night-shift to day-shift providers; of the changes made, 29% (12.7% of cases overall) were major changes. This study suggests that structured feedback on clinical reasoning for overnight admissions is a promising approach to improve residents' diagnostic calibration, particularly given how often diagnostic changes occur.

Complete Genome Sequences of BK Polyomavirus Strains from Two Patients with Urinary Tract Infection, Sequenced Using the Ion Torrent Platform.

BK polyomavirus is an important pathogen in kidney transplant patients. We report here two complete genome sequences, those of isolates CAMB-1035 and CAMB-1055, identified in two urine samples tested for urinary tract infection at a hospital in eastern England, United Kingdom. Variation and phylogenetic analyses indicate that both isolates belong to subtype Ib-1.

Antibiotic resistance potential of the healthy preterm infant gut microbiome.

BACKGROUND: Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. RESULTS: Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. CONCLUSIONS: We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing.

Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.

Challenges of the Unknown: Clinical Application of Microbial Metagenomics.

Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK.

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.