An Update on Myocarditis in Forensic Pathology.

In forensic medicine, myocarditis is a complicated topic in the context of sudden death and medical malpractice. A good knowledge of the etiopathology, histopathology, and available literature are both indispensable and essential for the correct management and evaluation of the causal link. Some agents, which are rarely lethal for humans, are not necessarily related to death from myocarditis, even if an infection in other organs such as the gastrointestinal tract is documented. The diagnosis of the causes of death is often difficult and confusing. In some cases, the hypothetical diagnosis of myocarditis as the cause of death is formulated by deduction, causing error and misleading the correct temporal evaluation of pathological events. We reviewed the literature realizing that histomorphological data are scarce and often poorly documented. Only after COVID-19 have the histomorphological aspects of myocarditis been better documented. This is due to poor autopsy practice and poor accuracy in identifying the specific histotype of myocarditis with identification of the responsible agent. We believe that four points are essential for a better understanding and complete diagnosis of the disease: (1) clinical classification of myocarditis; (2) etiological classification of myocarditis; (3) pathophysiology of viral and bacterial infections with host response; and (4) histopathological diagnosis with precise identification of the histotype and pathogen. In the review we provide histological images from authoritative scientific references with the aim of providing useful information and food for thought to readers.

Microplastics and Nanoplastics in Atheromas and Cardiovascular Events.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging as a potential risk factor for cardiovascular disease in preclinical studies. Direct evidence that this risk extends to humans is lacking. METHODS: We conducted a prospective, multicenter, observational study involving patients who were undergoing carotid endarterectomy for asymptomatic carotid artery disease. The excised carotid plaque specimens were analyzed for the presence of MNPs with the use of pyrolysis-gas chromatography-mass spectrometry, stable isotope analysis, and electron microscopy. Inflammatory biomarkers were assessed with enzyme-linked immunosorbent assay and immunohistochemical assay. The primary end point was a composite of myocardial infarction, stroke, or death from any cause among patients who had evidence of MNPs in plaque as compared with patients with plaque that showed no evidence of MNPs. RESULTS: A total of 304 patients were enrolled in the study, and 257 completed a mean (±SD) follow-up of 33.7±6.9 months. Polyethylene was detected in carotid artery plaque of 150 patients (58.4%), with a mean level of 21.7±24.5 µg per milligram of plaque; 31 patients (12.1%) also had measurable amounts of polyvinyl chloride, with a mean level of 5.2±2.4 µg per milligram of plaque. Electron microscopy revealed visible, jagged-edged foreign particles among plaque macrophages and scattered in the external debris. Radiographic examination showed that some of these particles included chlorine. Patients in whom MNPs were detected within the atheroma were at higher risk for a primary end-point event than those in whom these substances were not detected (hazard ratio, 4.53; 95% confidence interval, 2.00 to 10.27; P<0.001). CONCLUSIONS: In this study, patients with carotid artery plaque in which MNPs were detected had a higher risk of a composite of myocardial infarction, stroke, or death from any cause at 34 months of follow-up than those in whom MNPs were not detected. (Funded by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale and others; ClinicalTrials.gov number, NCT05900947.).

COVID-19: detection methods in post-mortem samples.

COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed paraffin-embedded (FFPE) autopsy tissues could help to underlie mechanisms of the pathogenesis that are not well understood.The gold standard for COVID-19 detection in FFPE samples remains the qRT-PCR as in swab samples, contextually other methods have been developed, including immunohistochemistry (IHC), and in situ hybridization (ISH). In this manuscript, we summarize the main data regarding the methods of COVID-19 detection in pulmonary and extra-pulmonary post-mortem samples, and especially the sensitivity and specificity of these assays will be discussed.

Long-Term Prognosis among COVID-19 Patients: The Predictive Role Played by Hyperinflammation and Arrhythmic Disorders in Fatal Outcome.

Limited data are available on outcomes among COVID-19 patients beyond the acute phase of the disease. All-cause mortality among our COVID-19 patients one year after hospital discharge and factors/conditions associated with death were evaluated. All patients discharged from our COVID center were periodically evaluated by clinical assessment and by digital healthcare registry consultation. All findings acquired on discharge day represented the baseline data and were utilized for statistics. Of the 208 patients admitted, 187 patients were discharged. Among these, 17 patients died within 12 months (non-survivors). Compared to survivors, non-survivor patients were significantly (p < 0.05) older, exhibited significantly greater comorbidities and prevalence of active malignancy, heart failure, and arrhythmias, and showed significantly higher circulating levels of B-type natriuretic peptide, troponin, C-reactive protein, and d-dimer, as well as a longer heart-rate-corrected QT interval and significantly lower values for the glomerular filtration rate. Following multivariate analysis, cancer, arrhythmias, and high C-reactive protein levels were found to be factors independently associated with death. At the one-year follow-up, about 9% of patients discharged from our COVID center had a fatal outcome. Ageing, myocardial injury, impaired renal function, and, in particular, cancer, hyperinflammation, and arrhythmias represented strong predictors of the worst long-term outcome among COVID-19 patients.

The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3.

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(-) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19.

PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte.

RATIONALE: About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. OBJECTIVE: To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. METHODS AND RESULTS: We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. CONCLUSIONS: The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.