Understanding yellow fever-associated myocardial injury: an autopsy study

Abstract

Background: Yellow fever (YF) is a viral hemorrhagic fever, endemic in parts of South America and Africa. There is scarce evidence about the pathogenesis of the myocardial injury. The objective of this study is to evaluate the cardiac pathology in fatal cases of YF.

Methods: This retrospective autopsy study included cases from the São Paulo (Brazil) epidemic of 2017-2019. We reviewed medical records and performed cardiac tissue histopathological evaluation, electron microscopy, immunohistochemical assays, RT-qPCR for YF virus (YFV)-RNA, and proteomics analysis on inflammatory and endothelial biomarkers.

Findings: Seventy-three confirmed YF cases with a median age of 48 (34-60) years were included. We observed myocardial fibrosis in 68 (93.2%) patients; cardiomyocyte hypertrophy in 68 (93.2%); endothelial alterations in 67 (91.8%); fiber necrosis in 50 (68.5%); viral myocarditis in 9 (12.3%); and secondary myocarditis in 5 (6.8%). Four out of five patients with 17DD vaccine-associated viscerotropic disease presented with myocarditis. The cardiac conduction system showed edema, hemorrhages and endothelial fibrinoid necrosis. Immunohistochemistry detected CD68-positive inflammatory interstitial cells and YFV antigens in endothelial and inflammatory cells. YFV-RNA was detected positive in 95.7% of the cardiac samples. The proteomics analysis demonstrated that YF patients had higher levels of multiple inflammatory and endothelial biomarkers in comparison to cardiovascular controls, and higher levels of interferon gamma-induced protein 10 (IP-10) in comparison to sepsis (p = 0.01) and cardiovascular controls (p < 0.001) in Dunn test.

Interpretation: Myocardial injury is frequent in severe YF, due to multifactorial mechanisms, including direct YFV-mediated damage, endothelial cell injury, and inflammatory response, with a possible prominent role for IP-10.

Funding: This study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, Bill and Melinda Gates Foundation, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

Keywords: 17DD; Autopsy; Chemokines; Cytokines; Heart conduction system; IP-10; Myocarditis; Vascular endothelium; YEL-AVD; Yellow fever.

Fig. 1

Histopathological cardiac findings in autopsy cases of severe yellow fever. a. Myocardial interstitial mononuclear inflammatory infiltrate and associated edema. b. Myocarditis, with mixed inflammatory infiltrate damaging cardiac fibers with dystrophic calcification (arrow). c. Secondary myocarditis due to Gram-negative bacilli, with hemorrhagic vascular necrosis (arrow). d. Myocarditis secondary to Gram-positive cocci colonies (arrow). e. Secondary myocarditis due to hyphomycetes with morphology compatible with Aspergillus spp (arrows). f. Epicarditis due to mixed inflammatory infiltrate. g. Hyaline necrosis, in a contraction band of cardiac fibers (arrow), fiber myolysis (inset), mononuclear inflammatory infiltrate and interstitial edema. h. Myocardial artery with tumefaction of endothelial cells, and fibrin within vascular lumen (arrow), perivascular mononuclear inflammatory infiltrate and edema. i. Epicardial venule with fibrinoid necrosis of the endothelium. j. Hemorrhage in the subendocardium and myocardium. Yellow fever subtype: a–g, i and j: wild-type; h: yellow fever vaccine-associated viscerotropic disease. Hematoxylin-Eosin. Magnification: C, F, J:100x; a, b, d, g, and i: 200x; e and h: 400x. Scale bars: 20 μm (c–e, h, i); 50 μm (a, b, g, j).

Fig. 2

Ultrastructural cardiac findings in autopsy cases of severe yellow fever. a. Disarrangement of sarcomeres and mitochondrias in the cardiomyocytes. b. Mitochondriosis and rupture of mitochondrial cristae. c. Dissociated collagen fibers due to perivascular edema. d. Myocardial endothelial cells with swollen cytoplasm, displaying pseudopods (arrow). e. Cytoplasm of endothelial cells showing vesicular degeneration of the smooth endoplasmic reticulum (black arrows), with some vesicles showing round electron-dense virus-like particles, measuring 110–150 nm (red arrows). f. Endothelial cell cytoplasm showing endoplasmic reticulum vesicle, containing two electron-dense virus-like particles, measuring between 80 and 90 nm (arrow). All images are from wild-type yellow fever cases. Scale bars: 500 nm (f); 1 μm (e); 2 μm (b, d); 10 μm (a).

Fig. 3

Immunohistochemistry findings in myocardial tissue of autopsy cases of severe yellow fever. a–g. Immunostaining of yellow fever virus antigen: within the cytoplasm of endothelial cells from myocardial vessels (a and b, arrows); within inflammatory cells (c, red arrow) and endothelial cells (c, black arrow) in focal mixed myocarditis, attributed to yellow fever; in circulating leukocytes, in a case with detectable YFV-RNA in the blood on the day of death (d, arrow); in perivascular inflammatory cells in the epicardium (e, arrow); in inflammatory cells among cardiac fibers (f and g, arrows). h–m. Myocardial inflammatory cell immunophenotype: CD68+ cells (h); CD45+ cells (i); NK (j) cells; T-CD4+ cells (k); T-CD8+ cells in a wild-type yellow fever case (l); and T-CD8+ cells in a case with acute viscerotropic disease associated with yellow fever vaccine and myocarditis (m). n. Increased angiopoietin-2 expression in venous (black arrow) and arterial (red arrow) vessels in cases of severe yellow fever. Yellow fever subtype: a–l, n: wild-type; m: yellow fever vaccine-associated viscerotropic disease. Peroxidase. Magnification: h, i, j, m, n: 100x; a, c, d, g, k, l: 200x; b, e and f: 400x. Scale bars: 5 μm (f); 20 μm (a–d, g, k); 50 μm (h–j, m, n).

Fig. 4

Pathology findings of the cardiac conduction system in autopsy cases of severe yellow fever. a. Macroscopy of the heart demonstrates a common finding in cases of severe yellow fever: subendocardial hemorrhage on the basal left part of the interventricular septum, in the topography of the conduction system (left bundle branch). b. Sinoatrial nodal artery (arrow) with endothelial swelling, fibrin aggregates within vascular lumen, edema of the vessel wall, and perivascular interstitial edema. c. Artery close to the sinoatrial node showing mycotic thrombus (arrows), in case of secondary infection by Aspergillus spp. d. Edema and interstitial hemorrhage (arrows) around the compact node (asterisk). e. Interstitial edema and hemorrhage (arrow) around the bundle of His (asterisk). f. Edema and interstitial hemorrhage (arrow) around the proximal portion of the left bundle branch. g. Edema and interstitial hemorrhage (arrow) around the distal portion of the left bundle branch. h and i. Immunostaining of yellow fever virus antigen in the cytoplasm of interstitial macrophages close to the sinoatrial node (h, arrow and inset) and within endothelial cells of some vessels in the myocardium (i, arrow), close to the bundle of His and left bundle branch. j. Increased angiopoietin-2 expression in vessels close to the sinoatrial node. k. Inflammatory infiltrate by activated macrophages (CD68+) around the sinoatrial nodal artery. l. Immunostaining for CD45 in interstitial lymphocytes (arrows), close to the bundle of His. All images are from wild-type yellow fever cases. Hematoxylin-Eosin: b–d, g; Masson’s trichrome: e, f; Peroxidase: h–l. Magnification: b, d–g: 100x; c, j–l: 200x; h, i: 400x. Scale bars: 20 μm (h, i); 50 μm (c, j–l); 100 μm (b, d–f, g).

Fig. 5

Tissue proteomics analysis of cytokines and endothelial biomarkers. Cytokines and endothelial lesion biomarkers dosages performed through multiplex immunoassay on fragments of frozen myocardial tissue obtained from autopsy from 69 patients with yellow fever, 6 with cardiovascular death and 6 with sepsis. All biomarkers dosages were normalized by total protein of each sample and only comparative data are shown. The Kruskal–Wallis test was used for comparisons between the 3 groups. When there was statistical significance, Dunn’s post-hoc test with Benjamini-Hochberg correction was used for multiple comparisons. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns: not significant. TNF: tumor necrosis factor; IFN-γ: Interferon-gama: interferon, IL: interleukin; MCP-1: monocyte chemoattractant protein 1; MIP1-β: macrophage inflammatory protein 1–beta; IP-10: Interferon gamma-induced protein 10; GM-CSF: granulocyte and macrophage colony stimulating factor; VEGF: vascular endothelial growth factor; VCAM-1: vascular cell adhesion molecule 1; ICAM-1: Intercellular adhesion molecule 1; PAI-1: plasminogen activation inhibitor; vWF-A2: von Willebrand factor A2.