Differential Deep RNA Sequencing for Diagnostic Detection of Microbial Infections in Inflammatory Cardiomyopathy.

BACKGROUND: Inflammatory heart disease can be triggered by a variety of causes, both infectious and noninfectious in nature. We hypothesized that inflammatory cardiomyopathy is potentially related to microbial infection. METHODS: In this retrospective study, we used deep RNA sequencing on formalin-fixed paraffin-embedded heart tissue specimens to detect pathogenic agents. We first investigated 4 single-sample cases to test the feasibility of this diagnostic protocol and further 3 control-sample paired cases to improve the protocol with differential metatranscriptomics next-generation sequencing (mtNGS) analysis. RESULTS: We demonstrate that differential mtNGS allows identification of various microbials as potentially pathogenic, for example, Cutibacterium acnes, Corynebacterium aurimucosum, and Pseudomonas denitrificans, which are usually commensal in healthy individuals. Differential mtNGS also allows characterization of human host response in each individual by profiling alterations of gene expression, networked pathways, and inferred immune cell compositions, information of which is beneficial for us to understand different etiologies and immunity roles in each case. Additionally, differential mtNGS allows the identification of genetic variants in patients that may contribute to their susceptibility to particular microbial infections. CONCLUSIONS: The demonstrated power of differential mtNGS in simultaneous capture of both the infectious microbial(s) and the status of human host immune response could help us better understand the pathogenesis of complex inflammatory cardiomyopathy, if conducted on a larger scale of the population. The developed differential mtNGS method could also shed light on its translation and adoption of such a laboratory test in clinic practice, allowing for a more effective diagnosis to guide therapeutic treatment of the disease.

Measuring the Efficacy of Pathology Career Recruitment Strategies in US Medical Students.

CONTEXT.­: Multiple articles and surveys in the literature suggest that medical students find a career in pathology undesirable and believe it is disproportionately focused primarily on the autopsy. OBJECTIVE.­: To measure the effect of applied interventions on medical student attitudes about the field of pathology. DESIGN.­: This prospective study involving medical students from first through fourth year was conducted as a pilot study in 2 medical schools in the United States. A 2-part anonymous survey regarding interest in pathology as a career and familiarity with the specialty using a 10-point scale was given to first- and second-year medical students before and after they listened to a 10-minute pathology career presentation. The same survey was given to third- and fourth-year medical students before and after a 4-week pathology elective. RESULTS.­: A total of 121 and 83 students responded to the survey before and after the intervention, respectively. Of the 121 students who responded to the survey before the intervention, 106 (87.6%) had not spent significant time in a pathology laboratory before the intervention. The majority of responses in interest in career, job responsibilities, and features of pathologists before and after the intervention demonstrated a statistically significant difference (P < .001). We compared survey scores of presentation versus 4-week rotation groups before and after the intervention. Students who experienced the presentation did not differ from students who experienced the rotation in the majority of questions related to interest in career, job responsibilities, and features of pathologists. CONCLUSIONS.­: Our study suggests that pathology exposure strategies can have a beneficial effect on student perceptions of the field and consideration of a career in pathology. Overall, the presentation intervention seemed to have the greatest effect on the first- and second-year students.

Inflammatory Cardiomyopathy: Case-based Review on Clinical Presentation, Diagnosis, and Management.

Inflammatory cardiomyopathy is a broad term encompassing any disease leading to myocardial inflammation with associated cardiac dysfunction. While endomyocardial biopsy remains the gold standard for diagnosis, noninvasive imaging techniques, such as cardiac magnetic resonance imaging and positron emission tomography, have become powerful tools to facilitate the identification of underlying myocardial inflammation. This review presents a series of clinical cases with some common etiologies of inflammatory cardiomyopathy, including diagnosis and management.

Lyme disease: a case report of a 17-year-old male with fatal Lyme carditis.

Lyme disease is a systemic infection commonly found in the northeastern, mid-Atlantic, and north-central regions of the United States. Of the many systemic manifestations of Lyme disease, cardiac involvement is uncommon and rarely causes mortality. We describe a case of a 17-year-old adolescent who died unexpectedly after a 3-week viral-like syndrome. Postmortem examination was remarkable for diffuse pancarditis characterized by extensive infiltrates of lymphocytes and focal interstitial fibrosis. In the cardiac tissue, Borrelia burgdorferi was identified via special stains, immunohistochemistry, and polymerase chain reaction. The findings support B. burgdorferi as the causative agent for his fulminant carditis and that the patient suffered fatal Lyme carditis. Usually, Lyme carditis is associated with conduction disturbances and is a treatable condition. Nevertheless, few cases of mortality have been reported in the literature. Here, we report a rare example of fatal Lyme carditis in an unsuspected patient.

Increased expression of oxidation-specific epitopes and apoptosis are associated with haptoglobin genotype: possible implications for plaque progression in human atherosclerosis.

OBJECTIVES: The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype. BACKGROUND: Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions. METHODS: Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively. RESULTS: In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p < 0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p < 0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p < 0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p < 0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p < 0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p < 0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p < 0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p < 0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p < 0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p < 0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p < 0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p < 0.003). CONCLUSIONS: These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

Inflammation, neovascularization and intra-plaque hemorrhage are associated with increased reparative collagen content: implication for plaque progression in diabetic atherosclerosis.

Sustained inflammation may stimulate a reparative process increasing early reparative type III collagen synthesis, promoting atherosclerotic plaque progression. We evaluated inflammation, neovascularization, intra-plaque hemorrhage (IPH), and collagen deposition in human aortic atherosclerotic plaques from patients with and without diabetes mellitus (DM). Plaques were procured at autopsy from lower thoracic and abdominal aorta from DM (n = 20) and non-DM (n = 22) patients. Inflammation and neovascularization were quantified by double-label immunochemistry and the IPH grade was scored using H&E-stained sections. Type I and type III collagens were quantified using Picro-Sirius red stain with polarization microscopy and computerized planimetry. In non-DM plaques, 27%, 40%, and 33% had mild, moderate and severe inflammation in the fibrous cap and shoulder compared with 2%, 30% and 68% in DM plaques (p < 0.001). The geometric mean neovessel count was increased in DM versus non-DM plaques (140 [95% CI: 119-165] versus 59 [95% CI: 51-70]; p < 0.001). The IPH grade was increased in DM verses non-DM plaques (0.82 ± 0.11 versus 0.29 ± 0.11; p < 0.001) (percentage grade). The density of type III was increased in DM plaques (0.16 ± 0.01 versus 0.06 ± 0.01; p < 0.001) with a non-significant reduction in type I density in DM when compared with non-DM (0.28 ± 0.03 versus 0.33 ± 0.03; p = 0.303) (content per mm²). The increase in type III collagen content correlated with total neovessel content (r = 0.58; p < 0.001) in DM plaques. In conclusion, our study suggests that enhanced type III collagen deposition was associated with inflammation, neovascularization and IPH, and may be a contributing factor in DM plaque progression.

Increased macrophage infiltration and neovascularization in congenital bicuspid aortic valve stenosis.

OBJECTIVES: Patients with congenital bicuspid aortic valves have aortic valve stenosis at a relatively young age compared with patients with tricuspid aortic valves. We hypothesize that aortic valve stenosis evolves from a more aggressive inflammatory process, with increased macrophage/T-cell and neovessel content in congenital bicuspid aortic valveswhen compared with that seen in tricuspid valves. METHODS: Fifty-one severely stenotic aortic valves were obtained at the time of aortic valve replacement. A total of 17 bicuspid and 34 tricuspid aortic valves were evaluated. Macrophage/T-cell infiltration (CD68 plus CD3) and neovessel density (CD34) were evaluated with immunohistochemical staining. Leaflet calcification and ossification were also quantified. Real-time polymerase chain reaction was used to assess expression of chondromodulin 1 and vascular endothelial growth factor. RESULTS: The density of macrophages/T cells was greater in congenital bicuspid aortic valves than in tricuspid valves (51 ± 31 vs 23 ± 13 cells/mm(2), P = .002). Neovascularization was more frequently noted in congenital bicuspid aortic valves when compared with tricuspid valves (31 ± 10 vs 21 ± 9 vessels/mm(2), P = .0005), and calcification was more severe (P = .03). Expression of chondromodulin 1 demonstrated a 6-fold downregulation (P = .0003) and expression of vascular endothelial growth factor demonstrated a 2-fold increase (P = .02) in congenital bicuspid aortic valves compared with that seen in tricuspid valves. Multivariable analyses demonstrated significant associations between bicuspid aortic valve anatomy and increased inflammatory cell infiltration (ß = 25.8, P = .0007) and neovascularization (ß = 9.4, P = .001), despite adjusting for measured covariates. CONCLUSIONS: The pathogenesis of aortic valve stenosis in bicuspid aortic valves is associated with a more aggressive inflammatory process with increased macrophage infiltration and neovascularization when compared with that seen in tricuspid valves.

Ischemic bowel due to embolization from an isolated mobile thrombus of the ascending aorta: a case report and review of the literature.

Aortic thrombi are commonly present in atherosclerotic and aneurysmatic aortas. Thrombus formation in an aorta with or focal atherosclerosis in a patient without risk factors is rare. A 63-year-old woman with dementia and hypothyroidism presented with hypotension and respiratory distress. Work-up revealed leukocytosis, sinus tachycardia, and proximal small bowel obstruction. At emergent laparotomy, a superior mesenteric artery thomboembolus was identified with necrosis of surrounding bowel. The patient expired on hospital day five. Autopsy revealed a 1.4 cm thrombus overlying an isolated atherosclerotic plaque in the ascending aorta and infarctions of the spleen, liver, and right kidney as well as occlusive thromboembolism of the superior mesenteric artery. This case report illustrates lethal complications from an unsuspected aortic thrombus. Work-up for patients presenting with signs of peripheral embolization, or in this case, necrotic bowel, should include the aorta as a source of embolic thrombi.

Clinical setting and extent of premortem evaluation do not predict autopsy discrepancy rates.

Autopsy rates have been affected by a number of factors, including technological advances and clinician beliefs of the diminished value of the autopsy. Such factors have resulted in a cultural shift in medicine away from the autopsy. Despite this shift, a number of studies have shown significant differences between antemortem clinical diagnoses and postmortem findings. Surveys of clinician beliefs about the autopsy have pointed toward antemortem diagnostic advancements as an important factor in declining rates. No study to date has addressed the hypothesis that such perceptions in diagnostic certainty have been matched by an actual decay in the yield of valuable or new information obtained by the autopsy. To address this hypothesis, we retrospectively compared the class I and class II discrepancies identified in 284 patients who died in three clinical settings with differing intensities of antemortem diagnostic workup. Despite a significantly different intensity of antemortem workup for patients in each clinical setting, including patients on a medical intensive care unit, patients on a surgical service and patients in an affiliated nursing home, discrepancy rates were found to be similar. Overall discrepancy rates for the medical intensive care unit, surgery service and nursing home patients were 27.8, 32.7 and 31.3%, respectively (P=0.84). In addition, we found no statistical difference in the complexity of workup in discrepant and nondiscrepant cases in each clinical setting. Our study data refute the hypothesis that the intensity of antemortem diagnostic evaluation correlates with an actual decrease in the rate of major diagnostic discrepancies identified at autopsy.

Haptoglobin genotype is a major determinant of the amount of iron in the human atherosclerotic plaque.

OBJECTIVES: We sought to test the hypothesis that haptoglobin (Hp) genotype is a determinant of the amount of iron in the atherosclerotic plaque. BACKGROUND: In atherosclerotic lesions, intraplaque hemorrhage releases free hemoglobin (Hb), whose incorporated iron can act as an oxidant and inflammatory stimulus. These effects are antagonized by Hp, which binds free Hb and facilitates its clearance from the plaque. The Hp gene has 2 alleles (1 and 2), giving rise to 3 genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. We previously hypothesized that Hp 2-2 individuals with diabetes mellitus (DM) have impaired clearance of Hb and its iron cargo from the plaque. METHODS: We identified the presence or absence of Perl's iron stain in 189 plaques obtained from 37 decedents at autopsy. RESULTS: Among DM, the prevalence of Perl's iron stain was increased in Hp 2-2 compared with that seen in Hp 1-1 or 2-1 (46.2% vs. 11.8%). After accounting for the within-decedent correlation of plaques, the prevalence ratio of Perl's iron stain associated with Hp 2-2 was 3.97 (95% confidence interval: 1.38 to 11.5; p = 0.025). In non-DM plaques, there was a nonsignificant trend towards a higher prevalence of iron staining in Hp 2-2 compared with that in Hp 1-1 or 2-1 (26.8% vs. 11.1%; prevalence ratio =2.40 [95% confidence interval: 0.81 to 7.09]; p = 0.114). CONCLUSIONS: These data support an impaired clearance of Hb from plaques in Hp 2-2 individuals, particularly in DM. The higher prevalence of plaque iron in Hp 2-2 DM individuals may contribute to the increased incidence of atherothrombotic events in these patients.

Human immunodeficiency virus (HIV) infects human arterial smooth muscle cells in vivo and in vitro: implications for the pathogenesis of HIV-mediated vascular disease.

Human immunodeficiency virus (HIV) infection is associated with accelerated atherosclerosis and vasculopathy, although the mechanisms underlying these findings have not been determined. Hypotheses for these observations include: 1) an increase in the prevalence of established cardiac risk factors observed in HIV-infected individuals who are currently experiencing longer life expectancies; 2) the dyslipidemia reported with certain HIV anti-retroviral therapies; and/or 3) the proinflammatory effects of infiltrating HIV-infected monocytes/macrophages. An unexplored possibility is whether HIV itself can infect vascular smooth muscle cells (SMCs) and, by doing so, whether SMCs can accelerate vascular disease. Our studies demonstrate that human SMCs can be infected with HIV both in vivo and in vitro. The HIV protein p24 was detected by fluorescence confocal microscopy in SMCs from tissue sections of human atherosclerotic plaques obtained from HIV-infected individuals. Human SMCs could also be infected in vitro with HIV by a mechanism dependent on CD4, the chemokine receptors CXCR4 or CCR5, and endocytosis, resulting in a marked increase in SMC secretion of the chemokine CCL2/MCP-1, which has been previously shown to be a critical mediator of atherosclerosis. In addition, SMC proliferation appeared concentric to the vessel lumen, and minimal inflammation was detected, unlike typical atherosclerosis. Our data suggest that direct infection of human arterial SMCs by HIV represents a potential mechanism in a multifactorial paradigm to explain the exacerbated atherosclerosis and vasculopathy reported in individuals infected with HIV.

Fibrous pleural tumor with hypoglycemia: case study.

Many neoplastic tumors exhibit paraneoplastic syndromes manifested by endocrinopathy. This is particularly true of intrathoracic tumors such as lung cancers, thymomas, carcinoid tumors and mediastinal germ cell neoplasm. Fibrous tumors of the pleura are rare intrathoracic tumors, which are usually benign and often grow to huge size. A subset of these neoplasms present with the syndrome of hypoglycemia. Although first reported more than 70 years ago, the diagnosis is rarely considered when a patient presents with syncope and hypoglycemia. This article reports a patient who presented with a large pleural mass and a hypoglycemic syndrome. (The disease was surgically cured.) The probable mechanism of hypoglycemia is discussed.

Giant blastoconidia of Candida albicans. A case report and review of the literature.

We describe a patient with extranodal non-Hodgkin lymphoma who developed systemic candidiasis after treatment with a cyclophosphamide-based chemotherapy regimen. Histologically, the fungal organisms demonstrated markedly enlarged blastoconidia with a variety of morphologic forms, mimicking other mycotic organisms, such as Cryptococcus neoformans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. The in vivo occurrence of such giant forms is rare, and when observed histologically may result in an erroneous diagnosis or a diagnosis of multiple mycotic organisms.

Nocardiosis presenting as an anterior mediastinal mass in a patient with sarcoidosis.

We report a patient with tissue-proven sarcoidosis receiving adrenocorticosteroid medication, who developed an enlarging mediastinal mass. Transcutaneous needle biopsy of the mass yielded pus which grew Nocardia asteroides on culture. Pleural effusion, bronchoesophageal fistula and brain nocardia metastases occurred. All evidence of active infection cleared with sulfa therapy. An enlarging mass in a patient with sarcoidosis unresponsive to corticosteroid therapy should provoke studies for other causes of mediastinal disease, including opportunistic infections.