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Supplementary Figure 3 from Vascular Endothelial Growth Factor-C Induces Lymphangitic Carcinomatosis, an Extremely Aggressive Form of Lung Metastases

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Supplementary Figure 2 from Vascular Endothelial Growth Factor-C Induces Lymphangitic Carcinomatosis, an Extremely Aggressive Form of Lung Metastases

Supplementary Figure 4 from Vascular Endothelial Growth Factor-C Induces Lymphangitic Carcinomatosis, an Extremely Aggressive Form of Lung Metastases

Histological examination of endomyocardial biopsy or myocardium at autopsy is key to the diagnosis of myocarditis. Among pathologists there is currently extensive variability in routine practice and criteria used to define, diagnose and report myocarditis as well as to achieve consensus on cases. Two manuscripts emphasizing the need to standardize and implement histopathological diagnostic criteria for myocarditis are reviewed.

Sudden cardiac death is, by definition, an unexpected, untimely death caused by a cardiac condition in a person with known or unknown heart disease. This major international public health problem accounts for approximately 15-20% of all deaths. Typically more common in older adults with acquired heart disease, SCD also can occur in the young where the cause is more likely to be a genetically transmitted process. As these inherited disease processes can affect multiple family members, it is critical that these deaths are appropriately and thoroughly investigated. Across the United States, SCD cases in those less than 40 years of age will often fall under medical examiner/coroner jurisdiction resulting in scene investigation, review of available medical records and a complete autopsy including toxicological and histological studies. To date, there have not been consistent or uniform guidelines for cardiac examination in these cases. In addition, many medical examiner/coroner offices are understaffed and/or underfunded, both of which may hamper specialized examinations or studies (e.g., molecular testing). Use of such guidelines by pathologists in cases of SCD in decedents aged 1-39 years of age could result in life-saving medical intervention for other family members. These recommendations also may provide support for underfunded offices to argue for the significance of this specialized testing. As cardiac examinations in the setting of SCD in the young fall under ME/C jurisdiction, this consensus paper has been developed with members of the Society of Cardiovascular Pathology working with cardiovascular pathology-trained, practicing forensic pathologists.

The two histopathology benchmarks used to diagnose myocarditis are the Dallas Criteria, developed in 1984 and the European Society of Cardiology criteria, developed in 2013, which added immunohistochemistry for the detection of CD3+ T cells (lymphocytes) and CD68+ macrophages. Despite their near universal acceptance, the extent to which pathologists use these criteria or their own criteria to consistently render the diagnosis of myocarditis on endomyocardial biopsy (EMB) is unknown. We digitally scanned slides from 100 heart biopsies, including a trichrome stain and immunostaining, that were chosen as representative of myocarditis, non-myocarditis, and borderline myocarditis, as diagnosed per one institution's use of the Dallas Criteria. Eight blinded international cardiovascular experts were asked to render diagnoses and offer a confidence score on each case. No clinical histories were shared. There was full initial agreement across all experts on 37 cases (16 myocarditis and 21 non-myocarditis) and moderate consensus on 35 cases. After individual inquiries and group discussion, consensus was reached on 90 cases. Diagnostic confidence was highest among the myocarditis diagnoses, lowest for borderline cases, and significantly different between the three diagnostic categories (myocarditis, borderline myocarditis, non-myocarditis; P-value=8.49 × 10

The heart oxidizes fatty acids for its energy production. The physiological balance between fatty acid uptake and its oxidation prevents lipid accumulation in cardiac myocytes. However, accumulation of lipids due to various processes such as obesity, diabetes, heart failure, myocardial ischemia or infarction can result in damage to the heart tissue, also known as cardiolipotoxicity. We present a unique case of a 69-year-old gentleman with a history of heart failure and ventricular tachycardia. Endomyocardial biopsy to assess for restrictive cardiomyopathy/amyloid showed no evidence of amyloid, significant inflammation or fibrosis, but did show intracellular accumulation of significant amorphous material in most cardiac myocytes. We review the literature regarding the pathogenesis of cardiolipotoxicity, which has no definite cause or treatment yet identified.

The endpoint in emergent management of acute massive pulmonary embolism (PE) has traditionally been with embolectomy through a standard median sternotomy. This approach is limited in both exposure and concomitant functional morbidity associated with sternotomy. In a previous publication, we described a novel minimally invasive, thoracoscopically assisted approach to pulmonary embolectomy. This approach utilized a small 5-cm left upper parasternal thoracotomy and femoral cardiopulmonary bypass to conduct thoracoscopically assisted surgical pulmonary embolectomy. The first publication featured three patients that had a massive pulmonary embolus that was treated with minimally invasive pulmonary embolectomy, and the initial data was positive and suggested that this approach is safe and feasible. We now broaden our experience with another two patients who underwent this approach, and highlight a number of technical and management modifications that have been made to optimize the procedure. These lessons learned will ideally benefit future surgeons as this approach is more heavily implemented in practice.

Introduction Renal cell carcinoma is often discovered at an early stage due to the increased use of imaging studies in the current era; therefore, its presentation as a gigantic renal cell carcinoma is rarely encountered. Case presentation A 59‐year‐old male presented to our hospital due to dizziness, fatigue, and increasing abdominal distension. A computed tomography scan showed an extremely large mass occupying most of the abdomen and pelvis. Surgical resection of the mass was performed. The largely cavitary mass with fibrous capsule was 43 cm and 13.0 kg, and contained a large amount of necrotic tissue. A portion of the left kidney was identified at the periphery of the mass, indicating that the tumor was arising from the left kidney. The final pathologic diagnosis was type 1 papillary renal cell carcinoma. Conclusion To the best of our knowledge, this tumor is the world’s largest malignant renal tumor.

Despite patients having increased access to their own electronic health record (EHR) in recent times, patients are often still not considered a primary audience of pathology reports. An alternative to in-person patient education is the use of multimedia programming to enhance health literacy. Curated video presentations designed to explain diagnosis-specific pathology terms were reviewed by a board-certified pathologist and oncologist team and then shown to patients with a primary diagnosis of either pancreatic, colorectal, or prostate cancer in-clinic; these patients then completed a secure electronic survey immediately afterwards. Seventy patients were surveyed, with 91% agreeing or strongly agreeing that the video they watched increased their understanding of the medical terms used in their pathology reports, with a corresponding average Likert score (ALS) of 4.21 (SD = 0.77, CI = ± 0.18). Furthermore, 95% agreed or strongly agreed that the video they watched both enhanced their understanding of the role of the pathologist in diagnosing cancer (ALS = 4.27; SD = 0.65, CI = ± 0.15) and reported they found the video useful (ALS = 4.27; SD = 0.53, CI = ± 0.13). Curated videos such as those utilized in this study have the potential to increase patient health literacy and inform patients of the multidisciplinary nature of cancer diagnosis.

Hypertrophic cardiomyopathy affects 0.5% of the population. Advanced testing is considered, including cardiac catheterization, endomyocardial biopsy, and cardiopulmonary exercise testing. Right and left heart catheterization provides essential hemodynamic data, identifies patients who might benefit from septal reduction therapy, and assesses for comorbidities. Pathologic analysis reveals ventricular hypertrophy, myocardial disarray, and endocardial and interstitial fibrosis. Routine endomyocardial biopsy is not recommended unless other conditions that cause hypertrophy need to be ruled out. Cardiopulmonary exercise testing provides useful physiologic data, allows monitoring of the response to medication and surgical interventions, estimates prognosis, and guides referral for orthotopic heart transplantation.

Enterovirus D68 (EV-D68) infection has been associated with outbreaks of severe respiratory illness and increased cases of nonpolio acute flaccid myelitis. The patterns of EV-D68 circulation and molecular epidemiology are not fully understood. In this study, nasopharyngeal (NP) specimens collected from patients in the Lower Hudson Valley, New York, from 2014 to 2018 were examined for rhinovirus/enterovirus (RhV/EV) by the FilmArray respiratory panel. Selected RhV/EV-positive NP specimens were analyzed using two EV-D68-specific real-time RT-PCR assays, Sanger sequencing and metatranscriptomic next-generation sequencing. A total of 2,398 NP specimens were examined. EV-D68 was detected in 348 patients with NP specimens collected in 2014 (n = 94), 2015 (n = 0), 2016 (n = 160), 2017 (n = 5), and 2018 (n = 89), demonstrating a biennial upsurge of EV-D68 infection in the study area. Ninety-one complete or nearly complete EV-D68 genome sequences were obtained. Genomic analysis of these EV-D68 strains revealed dynamics and evolution of circulating EV-D68 strains since 2014. The dominant EV-D68 strains causing the 2014 outbreak belonged to subclade B1, with a few belonging to subclade B2. New EV-D68 subclade B3 strains emerged in 2016 and continued in circulation in 2018. Clade D strains that are rarely detected in the United States also arose and spread in 2018. The establishment of distinct viral strains and their variable circulation patterns provide essential information for future surveillance, diagnosis, vaccine development, and prediction of EV-D68-associated disease prevalence and potential outbreaks.

Since publication of our initial experience with non-sternotomy minimally invasive pulmonary embolectomy (MIPE) via a left mini thoracotomy, we have expanded our experience, refined the operation and streamlined the post-operative management of patients. Our initial publication described three patients who underwent MIPE.1 We described our technique which included peripheral cardiopulmonary bypass (CPB) via femoral arterial and venous cannulation, left sided 5cm anterior thoracotomy in the 3rd intercostal space, identification and incision of the main pulmonary artery distal to the pulmonic valve, extraction of clot with subsequent primary closure of the pulmonary artery, and use of a 5mm, 30 degree laparoscope as an adjunct to assess clearance of the pulmonary artery.2 The patients included in this series had no post-operative complications, had a mean hospital length of stay of three days with mid-term follow-up up to 8-months revealing no untoward complications of the procedure. With early success of the MIPE at our institution, we began employing it preferentially over sternotomy with central CPB and pulmonary embolectomy. Since initial publication of our results, we have performed the MIPE in two additional patients with excellent outcomes. We herein present augmentations we’ve made to the procedure with a case-presentation which highlights these adaptations.

Background: Pulmonary complications often cause morbidity and mortality in pediatric allogeneic hematopoietic stem cell transplant (HSCT) recipients. While detection of infection and initiation of appropriate antimicrobial therapy improves survival, present techniques oftentimes do not detect infections in bronchoalveolar lavage (BAL) samples because of pretreatment with antimicrobial therapies and the need for a priori knowledge of likely viral pathogens, decreasing the yield of BAL. Objective: We evaluated whether RNA-based massively parallel sequencing (MPS) would improve detection of infections in BAL fluid in pediatric allogeneic HSCT recipients. Results: Nine patients underwent 10 BAL (1 patient underwent 2 BAL) and had sufficient BAL fluid for inclusion in this study. Clinical microbiological testing identified infections in 7 patients, and MPS identified infections in 5 patients, although some of these detected organisms were not detected by clinical testing. Results were fully concordant in 5 patients, fully discordant in 3 patients, and partially discordant in 2 patients. Bacterial, viral, and fungal infections were detected via both techniques. Conclusion: This suggests that MPS in conjunction with routine clinical testing increases the yield of detection of infectious organisms in the BAL fluid.

The surveillance of health care-associated infection (HAI) is an essential element of the infection control program. While whole-genome sequencing (WGS) has widely been adopted for genomic surveillance, its data processing remains to be improved. Here, we propose a three-level data processing pipeline for the precision genomic surveillance of microorganisms without prior knowledge: species identification, multi-locus sequence typing (MLST), and sub-MLST clustering. The former two are closely connected to what have widely been used in current clinical microbiology laboratories, whereas the latter one provides significantly improved resolution and accuracy in genomic surveillance. Comparing to a broadly used reference-dependent alignment/mapping method and an annotation-dependent pan-/core-genome analysis, we implemented our reference- and annotation-independent, k-mer-based, simplified workflow to a collection of Acinetobacter and Enterococcus clinical isolates for tests. By taking both single nucleotide variants and genomic structural changes into account, the optimized k-mer-based pipeline demonstrated a global view of bacterial population structure in a rapid manner and discriminated the relatedness between bacterial isolates in more detail and precision. The newly developed WGS data processing pipeline would facilitate WGS application to the precision genomic surveillance of HAI. In addition, the results from such a WGS-based analysis would be useful for the precision laboratory diagnosis of infectious microorganisms.

Atherosclerotic cardiovascular disease, a chronic inflammatory condition of multifactorial etiology, is the leading cause of death worldwide. The concept of ‘bacterial persistence’ has been proposed as one of several contributing factors to this disease. We hypothesized that the infectious agent(s) found at the site of the atheroma may be dormant but perpetuate virulence in response to host defense and other physiological triggers. In this study, we sought to identify the source of persistent infection in human atherosclerotic plaque and define how pathogen virulence and host defenses mediate plaque vulnerability. Whole genome sequencing (WGS) was used to identify bacteria from pure cultures obtained from atherosclerotic tissues of living subjects diagnosed with more than 70% occlusion of the carotid artery undergoing carotid endarterectomy (CE). WGS identified the predominant species as S. pasteuri (Spv) in all CE isolates grown in pure culture except one isolate which was identified as B. licheniformis. All S. pasteuri isolates (Spvs) were found to contain genes for widespread virulence, invasion, and intracellular resistance. As macrophages (Mφs) play a decisive role at all stages of this disease we treated mouse Mφs (RAW 264.7) with Spvs. While all Spvs tested demonstrated their ability to survive phagocytosis, the highly virulent Spvs also activated Mφs and induced trans-endothelial cell migration of these cells which was mediated via the CC chemokine CCL1 and its receptor CCR8. In conclusion, we show that Spvs are found in CE plaques, have the capacity to survive phagocytosis and induce the transmigration of Mφs across an endothelial barrier in a CCL1-CCR8 dependent process. These findings highlight the significance of carotid vessels as a reservoir for S. pasteuri, a pathogen found in products used routinely in human consumption; this may explain how microbial pathogenicity modulates plaque vulnerability in atherosclerosis.

Whole-genome sequencing is increasingly adopted in clinical settings to identify pathogen transmissions, though largely as a retrospective tool. Prospective monitoring, in which samples are continuously added and compared to previous samples, can generate more actionable information. To enable prospective pathogen comparison, genomic relatedness metrics based on single-nucleotide differences must be consistent across time, efficient to compute and reliable for a large variety of samples. The choice of genomic regions to compare, i.e., the core genome, is critical to obtain a good metric. We propose a novel core genome method that selects conserved sequences in the reference genome by comparing its k-mer content to that of publicly available genome assemblies. The conserved-sequence genome is sample set-independent, which enables prospective pathogen monitoring. Based on clinical data sets of 3436 S. aureus, 1362 K. pneumoniae and 348 E. faecium samples, ROC curves demonstrate that the conserved-sequence genome disambiguates same-patient samples better than a core genome consisting of conserved genes. The conserved-sequence genome confirms outbreak samples with high sensitivity: in a set of 2335 S. aureus samples, it correctly identifies 44 out of 44 known outbreak samples, whereas the conserved-gene method confirms 38 known outbreak samples.

Caveolin-1 (cav-1) has been shown to play a significant role in the pathogenesis of pulmonary hypertension (PH). In the monocrotaline model of PH, the loss of endothelial cav-1 as well as reciprocal activation of proliferative and anti-apoptotic pathways initiate the disease process and facilitate its progression. In order to examine the role of cav-1 in hypoxia-induced PH, we exposed rats and neonatal calves to hypobaric hypoxia and obtained hemodynamic data and assessed the expression of cav-1 and related proteins eNOS, HSP90, PTEN, gp130, PY-STAT3, β-catenin, and Glut1 in the lung tissue. Chronic hypoxic exposure in rats (48 h–4 weeks) and calves (two weeks) did not alter the expression of cav-1, HSP90, or eNOS. PTEN expression was significantly decreased accompanied by PY-STAT3 activation and increased expression of gp130, Glut1, and β-catenin in hypoxic animals. We also examined cav-1 expression in the lung sections from steers with chronic hypoxic disease (Brisket disease) and from patients with chronic lung disease who underwent lung biopsy for medical reasons. There was no cav-1 loss in Brisket disease. In chronic lung disease cases, endothelial cav-1 expression was present, albeit with less intense staining in some cases. In conclusion, hypoxia did not alter the cav-1 expression in experimental models. The presence of cav-1, however, did not suppress hypoxia-induced activation of PY-STAT3 and β catenin, increased gp130 and Glut1 expression, or prevent the PTEN loss, indicating cav-1 dysfunction in hypoxia-induced PH.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response.

We used 4 different bioinformatics algorithms to evaluate the application of a metagenomic shot-gun sequencing method in detection of Enterovirus D68 and other respiratory viruses in clinical specimens. Our data supported that next-generation sequencing, combined with improved bioinformatics tools, is practically feasible and useful for clinical diagnosis of viral infections.

Dialysis-related amyloidosis predominantly occurs in osteo-articular structures and dialysis-related amyloid (DRA) substances also deposit in extra-articular tissues. Clinical manifestations of DRA include odynophagia, gastrointestinal hemorrhage, intestinal obstruction, kidney stones, myocardial dysfunction, and subcutaneous tumors. The pathological characteristics of DRA in the heart of hemodialysis patients have rarely been reported. We report the case of a 73-year-old female with a history of cerebral palsy and end-stage renal disease status post two failed renal transplants who had been on hemodialysis for 30 years. The patient was admitted with the working diagnosis of pneumonia. An echocardiography showed markedly reduced biventricular function manifested by low blood pressure with systolic in the 70s and elevated pulmonary artery pressure of 45 mmHg, which did not respond to therapy. Following her demise, the autopsy revealed bilateral pulmonary edema and pleural effusions. There was cardiac amyloid deposition exclusively in the coronary arteries but not in the perimyocytic interstitium. Amyloids were also found in pulmonary and intrarenal arteries and the colon wall. Previous case reports showed that beta 2-microglobulin amyloid deposits in various visceral organs but less frequently in the atrial and/or the ventricular myocardium. In the present case, amyloids in the heart were present in the intramural coronary arteries causing myocardial ischemia and infarction, which was the immediate cause of death.

e15155 Background: Novel immunotherapies are becoming a viable option for advanced lung cancer treatment. High-throughput gene expression profiling (GEP) has enabled better understanding of patient responsiveness to targeted immunotherapy. However, non-invasive blood-based signatures are needed to monitor and predict response. Methods: To evaluate the predictive and prognostic role of blood-based GEP, we designed a longitudinal study by enrolling 15 patients with advanced lung cancers. A total of 65 samples were collected for RNA sequencing, ~4 blood specimens per patient, before and during anti-PD-1 treatment. We used multiple analyses, including time-course differential gene expression, principal component, lymphocyte compartment deconvolution, and genetic mutation, to search for and assess potential predictive and prognostic aspects. Results: Of 15 patients, 11 were classified as Responders (partial responders) and four were Non-Responders (one stable and three progressive diseases). Our analyses demonstrated: 1) By comparing baseline GEPs from Responders vs. Non-Responders before the first treatment, we identified potential markers (e.g., LY6E is significantly lower expressed in Responders, with Log2 Fold change = -3.44 and p = 1.83E-04) that can be used as predictors of responsiveness of the patients; 2) Immunoglobulin subunits- and T cell receptor complex-related genes were differentially expressed in Responders (DAVID analysis, p = 6.7E-3 and 2.1E-2, respectively), but not in Non-responders; 3) γδ T cells in the lymphocyte compartment were relatively increased in Responders; 4) Despite a different set of genes differentially expressed at different time points, the biggest GEP changes were at ~ week 6, after the second treatment. Additionally, we observed certain genes consistently up- or down-regulated through the whole course of treatment. Furthermore, after the first treatment, genes in the immune response pathway were regulated to different directions in Responders and Non-Responders. For example, interleukin receptor genes, such as IL18R1 and IL18RAP, and CD24 were down-regulated in Responders, but up-regulated in Non-Responders (p = 0.042, 0.023 and 0.044 respectively, t-test for the differential expression in these two groups). Conclusions: The utility of blood GEP to identify predictive and prognostic factors for precision immunotherapy is encouraging. Nevertheless, these results, predictive of the anti-PD-1 clinical response, are preliminary and need to be validated in a larger cohort.

Cardiac amyloidosis results from amyloid deposition into the extracellular space of the heart resulting in fibrosis, myocardial dysfunction, heart failure, and arrhythmias. Most common causes of amyloid affecting the heart include light chain (AL) or Transthyretin (TTR) amyloid. The aim of our study

Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone, and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC &beta, lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively, more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.

"Since the pathological conditions take place at the cellular level, viral myocarditis and postinfectious autoimmunity can be suggested but not diagnosed clinically. All clinical methods including imaging techniques are misleading if infectious agents are involved. Accurate diagnosis demands simultaneous histologic, immunohistochemical, and molecular biological workup of the tissue. If the primary infectious or immune-mediated causes of the disease are carefully defined by clinical and biopsy-based tools, specific antiviral treatment options in addition to basic symptomatic therapy are available under certain conditions. These may allow a tailored cause-specific treatment that improves symptoms and prognosis of patients with acute and chronic disease." Uwe Kühl; Heinz-Peter SchultheissViral myocarditis.Swiss Medical Weekly. 144():w14010, JAN 2014 DOI:10.4414/smw.2014.14010.

Hypertrophic cardiomyopathy affects 0.5% of the population. Advanced testing is considered, including cardiac catheterization, endomyocardial biopsy, and cardiopulmonary exercise testing. Right and left heart catheterization provides essential hemodynamic data, identifies patients who might benefit from septal reduction therapy, and assesses for comorbidities. Pathologic analysis reveals ventricular hypertrophy, myocardial disarray, and endocardial and interstitial fibrosis. Routine endomyocardial biopsy is not recommended unless other conditions that cause hypertrophy need to be ruled out. Cardiopulmonary exercise testing provides useful physiologic data, allows monitoring of the response to medication and surgical interventions, estimates prognosis, and guides referral for orthotopic heart transplantation.

Whole-genome sequencing is increasingly adopted in clinical settings to identify pathogen transmissions. Currently, such studies are performed largely retrospectively, but to be actionable they need to be carried out prospectively, in which samples are continuously added and compared to previous samples. To enable prospective pathogen comparison, genomic relatedness metrics based on single nucleotide differences must be consistent across time, efficient to compute and reliable for a large variety of samples. The choice of genomic regions to compare, i.e., the core genome, is critical to obtain a good metric.We propose a novel core genome method that selects conserved sequences in the reference genome by comparing its k-mer content to that of publicly available genome assemblies. The conserved-sequence genome is sample set-independent, which enables prospective pathogen monitoring. Based on clinical data sets of 3436 S. aureus, 1362 K. pneumoniae and 348 E. faecium samples, we show that the conserved-sequence genome disambiguates same-patient samples better than a core genome consisting of conserved genes. The conserved-sequence genome confirms outbreak samples with high accuracy: in a set of 2335 S. aureus samples, it correctly identifies 44 out of 45 outbreak samples, whereas the conserved gene method confirms 38 out of 45 outbreak samples.

Complete genome sequences of four toxigenic Clostridium difficile isolates from patients in the lower Hudson Valley, New York, USA, were achieved. These isolates represent four common sequence types (ST1, ST2, ST8, and ST42) belonging to two distinct phylogenetic clades. All isolates have a 4.0- to 4.2-Mb circular chromosome, and one carries a phage.

PRESENTATION Cardiogenic shock was the first sign of a devastating disorder in a 27-year-old woman with multiple medical problems. She presented to an outside hospital after 2 days of nausea, epigastric pain, and worsening dyspnea on exertion. Her medical history included systemic lupus erythematosus, autoimmune hepatitis, immune thrombocytopenia, autoimmune hemolytic anemia, attention deficit hyperactivity disorder, anxiety, and migraines. On physical examination, the patient appeared to be in severe respiratory distress. She was afebrile with blood pressure of 100/67 mm Hg, a heart rate of 116 beats per minute, and a respiratory rate of 30 breaths per minute with an oxygen saturation of 100% on supplemental oxygen delivered by a non-rebreather mask. She had no jugular venous distention. On cardiac auscultation, her heart rate was fast and regular with a holosystolic murmur, most prominent at the apex, and an S3 gallop was present. Rales were evident on auscultation of bilateral lung bases. Her extremities were cold with no edema. An electrocardiogram showed sinus tachycardia at 115 beats per minute, low voltage in the anterior leads, and nonspecific ST-T wave changes (Figure 1). Chest radiography revealed bilateral perihilar infiltrates compatible with pulmonary edema. A complete blood count and comprehensive metabolic panel disclosed leukocytosis (total white blood cell count, 42.1 10 cells/mm), anemia (hemoglobin, 10.6 g/dL), thrombocytopenia (platelet count, 135,000/mm), elevated aspartate

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

Xp11 (TFE3) translocation renal cell carcinoma (RCC) is officially recognized as a distinct subtype of RCC in the 2004 WHO classification. This neoplasm is characterized by several chromosomal translocations between the TFE3-involving Xp11.2 breakpoint and various fusion partners. To date, five partner genes have been identified, that is, PRCC in 1q21, PSF in 1q34, ASPL in 17q25, CLTC in 17q23, and NONO in Xq12; and three additional translocations have been reported with no partner gene being defined: t(X;3)(p11;q23), t(X;10)(p11;q23), and t(X;19)(p11;q13). Here, we report the identification of a novel TFE3 fusion partner, PARP14 in chromosome band3q21. We used RNA-seq on a 10-year-old FFPE (formalin-fixed, paraffin-embedded) tissue sample, which carried t(X;3)(p11;q23) as detected in the original cytogenetic study. The fusion transcript connected the 5'-end of the first two exons of PARP14 to the 3'-end of five exons of TFE3, which was verified by reverse transcription PCR (RT-PCR) and Sanger sequencing. Similar to other TFE3 fusions previously reported, the predicted PARP14-TFE3 product retains the nuclear localization and DNA-binding domains of TFE3. This finding expands the list of TFE3 translocation partner genes and re-emphasizes the essential oncogenic role of TFE3 fusion proteins in this tumor. Our result also clearly demonstrated the feasibility of identifying chromosomal translocation by RNA-seq in clinical FFPE, which are easily accessible and associated with valuable clinical information. © 2015 Wiley Periodicals, Inc.

Primary (localized) non-Hodgkin lymphoma (NHL) of the ovary is extremely rare; only a few cases have been reported in the literature. We report two cases of primary ovarian lymphoma (POL), one involving bilateral ovaries in a 15-year-old girl and other involving one ovary in a 5-year-old girl. This report describes detailed clinical, histopathological, and imaging findings, along with the review of literature of primary diffuse large B-cell lymphoma (DLBCL) arising from an ovary. In addition, we describe findings of targeted capture panel sequencing on both tumors and identify the major genetic mutations that are recurrently mutated in pan-cancers. Compared to the genomic mutation features of major subtypes of DLBCL, we distinguish that each POL belongs to distinctive subtypes, GCB (germinal center B-cell subtype) DLBCL and ABC (activated B-cell subtype) DLBCL, respectively. The findings from the genomic analysis may help to understand the pathogenesis of POL and to guide potential targeted therapy in the future.

Compared with conventional serologic, culture-based, and molecular-based diagnostic tests, next-generation sequencing (NGS) provides sequence-evidenced detection of various microbes, without prior knowledge, and thus is becoming a novel diagnostic approach. Herein we describe an RNA-based metatranscriptomic NGS (mtNGS) protocol for culture-independent detection of potential infectious pathogens, using clinical bronchoalveolar lavage specimens as an example. We present both an optimized workflow for experimental sequence data collection and a simplified pipeline for bioinformatics sequence data processing. As shown, the whole protocol takes approximately 24 to 36 hours to detect a wide range of Gram-positive and -negative bacteria and possibly other viral and/or fungal pathogens. In particular, we introduce a spike-in RNA mix as an internal control, which plays a critical role in mitigating false-positive and false-negative results of clinical diagnostic tests. Moreover, our mtNGS method can detect antibiotic resistance genes and virulence factors; although it may not be comprehensive, such information is imperative and helpful for the clinician to make better treatment decisions. Results from our preliminary testing suggest that the mtNGS approach is a useful alterative in diagnostic detection of emerging infectious pathogens in clinical laboratories. However, further improvements are needed to achieve better sensitivity and accuracy.

We report here the incidental detection and complete genome sequence of a urinary Escherichia coli strain harboring mcr-1 and resistant to colistin in a New York patient returning from Portugal in 2016. This strain, with sequence type 1485 (ST1485), was a non-extended-spectrum beta-lactamase (ESBL) and non-carbapenemase producer and carried the mcr-1 gene on an IncHI2 plasmid.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UC HL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis.

The extended-spectrum-β-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae , we identified three clinical isolates, CN1, CR14, and NY9, carrying both bla CTX-M and bla KPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, bla CTX-M and bla KPC were carried on two different plasmids. In contrast, CN1 had one copy of bla KPC-2 and three copies of bla CTX-M-15 integrated in the chromosome, for which the bla CTX-M-15 genes were linked to an insertion sequence, IS Ecp1 , whereas the bla KPC-2 gene was in the context of a Tn 4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn 4401a-bla KPC-2 -prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)- cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR- cas in K. pneumoniae strains and suggested that the evolving CRISPR- cas , with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of bla CTX-M and bla KPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae . Additionally, the implications from this study also raise concerns for the application of a CRISPR- cas strategy against antimicrobial resistance.

Background Since its introduction in 2009, use of daptomycin for treatment of enterococcal infections has resulted in the emergence of DNSE. Between 2009 and 2013, daptomycin nonsusceptibility among E. faecium was closely associated with emergence of a unique and dominant clone ST736 in our institution. In 2014, we instituted targeted measures to optimize the use of daptomycin. In this study, we describe the significant phenotypic and genotypic impact of reduced daptomycin use on clinical enterococcal isolates. Methods Enterococcal clinical isolates were recovered from January 2014 through December 2017. Daptomycin susceptibility was determined by MicroScan WalkAway™ System and confirmed by E-test. Selected DNSE and vancomycin-resistant E. faecium (VREfm) clinical isolates were analyzed by next-generation sequencing (NGS) using the Illumina systems to provide multilocus sequencing type (MLST). Daptomycin utilization data were extracted from pharmacy records. Results Targeted antibiotic stewardship initiatives consisted of preapproval, daily review for optimization of dose and duration, rapid de-escalation, consideration for appropriate alternative antibiotics for select disease syndromes and stopping of inappropriate daptomycin therapy. Over 4 years, this lead to a 39% reduction in overall use of daptomycin. Besides direct cost saving, this reduced use was associated with significant reduction in daptomycin nonsusceptibility from 12% to 4%, lowering of MIC90 from 8 to 4 μg/mL, and a clonal shift from dominant ST736 to ST117. Daptomycin Usage Days of Therapy /1,000 Days (Monthly Average) Phenotypic Changes Genotypic Changes MIC50 (μg/mL) MIC90 (μg/mL) % DNSE Dominant Genotype Among VREfm 2014 742 4 8 12 ST736 (38%) ST18 (21%) ST412 (12%) ST117 (11%) 2015 633 4 6 10 2016 576 4 4 7 2017 455 4 4 4 ST736 (5%) ST18 (4%) ST412 (11%) ST117 (41%) Conclusion A targeted antibiotic stewardship initiative to address rising rate of daptomycin nonsusceptibility among E. faecium, resulted in significant phenotypic and genotypic changes among clinical isolates. This study also shows successful integration of NGS in a clinical microbiology lab to validate phenotypic changes of daptomycin nonsusceptibility and to help design future infection control and antibiotic stewardship endeavors. Disclosures A. Dhand, Merck: Speaker’s Bureau, Speaker honorarium. Astellas: Scientific Advisor, Consulting fee.

Background Typical Infection Prevention to detect pathogen transmission in hospitals has relied on observation of (1) uncommon pathogen phenotypes or (2) greater than expected number of pathogen phenotypes in a given timeframe and/or location. Genome sequencing of targeted organisms in conjunction with routine patient geo-temporal information and antibiotic susceptibility data holds promise in identifying transmissions with greater sensitivity and specificity, saving time and effort in reviewing for transmission events. Methods In an on-going genomic sequencing surveillance effort in a tertiary care hospital, drug-resistant clinical isolates from the “ESKAPE” pathogens were routinely sequenced in 2017. In parallel, potential clusters were identified for 2017 through conventional Infection Prevention approaches. Groups identified by their genetic distances along with visualizations on antimicrobial susceptibilities, and patient location histories and dates were displayed in an interactive interface, Philips IntelliSpace Epidemiology (PIE), and reviewed by Infection Prevention. Results Among 656 patients, 1,239 drug-resistant ESKAPE samples were sequenced. Thirty-eight genetically related groups involving 196 patients were identified. Groups ranged in size from two to 44 patients, primarily consisting of VRE and MRSA. Notably, a review of the 38 groups identified 20 groups where the information at hand suggested a concern for transmission. 16 of the 20 were not previously identified by Infection Prevention. Using PIE to review all 38 groups identified from 1 year’s worth of data required 3 hours of time by an Infection Prevention professional, averaging less than 5 minutes per cluster, less than 1 minute per patient, and 11 minutes of review time per actionable opportunity. By conventional means, approximately 23 hours would have been required to review the genomic groups without the aid of the PIE tool. Conclusion The use of PIE’s genomic-defined groups, along with the integrated clinical data platform, allows for a greater ability, certainty, and speed to detect clusters of organisms representing transmission in the hospital setting. Applied prospectively, PIE can detect transmissions sooner than by conventional means for potential patient safety gains and cost savings. Disclosures D. Chen, Philips: Scientific Advisor, Consulting fee. M. Fortunato-Habib, Philips Healthcare: Collaborator and Employee, Salary. A. Hoss, Philips: Employee, Salary. R. Kolde, Philips: Employee, Salary. A. Dhand, Merck: Speaker’s Bureau, Speaker honorarium. Astellas: Scientific Advisor, Consulting fee. R. Sussner, Philips: Scientific Advisor, Consulting fee. J. Carmona, Philips Healthcare: Employee, Salary. B. Gross, Philips Healthcare: Employee, Investigator, Research Contractor, Scientific Advisor and Shareholder, Salary. J. Fallon, Philips Healthcare: Investigator, Research support.