Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem

Abstract

Background: Viral myocarditis is a major cause of sudden unexpected death in children and young adults. Until recently, coxsackievirus B3 (CVB3) has been the most commonly implicated virus in myocarditis. At present, no standard diagnosis is generally accepted due to the insensitivity of traditional diagnostic tests. This has prompted health professionals to seek new diagnostic approaches, which resulted in the emergence of new molecular pathological tests and a more detailed immunohistochemical and histopathological analysis. When supplemented with immunohistochemistry and molecular pathology, conventional histopathology may provide important clues regarding myocarditis underlying etiology.

Methods: This study is based on post-mortem samples from sudden unexpected death victims and controls who were investigated prospectively. Immunohistochemical investigations for the detection of the enteroviral capsid protein VP1 and the characterization and quantification of myocardial inflammatory reactions as well as molecular pathological methods for enteroviral genome detection were performed.

Results: Overall, 48 sudden unexpected death victims were enrolled. As for controls, 37 cases of unnatural traffic accident victims were studied. Enterovirus was detected in 6 sudden unexpected death cases (12.5 %). The control samples were completely enterovirus negative. Furthermore, the enteroviral capsid protein VP1 in the myocardium was detected in enterovirus-positive cases revealed by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike control samples, immunohistochemical investigations showed a significant presence of T and B lymphocytes in sudden unexpected death victims.

Conclusions: Our findings demonstrate clearly a higher prevalence of viral myocarditis in cases of sudden unexpected death compared to control subjects, suggesting that coxsackie B enterovirus may contribute to myocarditis pathogenesis significantly.

Figure 1

Histological specimen (hematoxylin-eosin staining) from Sudden Unexpected Death victims demonstrating active myocarditis. [1A, 1B, 1C] Areas of diffuse myocardial necrosis with large inflammatory infiltrates (arrows). [1D]Control samples (unnatural traffic accident deaths) showing no significant pathological findings.

Figure 2

Immunohistochemical detection of enteroviral capsid protein VP1 in post-mortem myocardial samples. [2A, 2B] Enteroviral capsid protein VP1 detected inside myocytes (arrows) suggesting a confluent invasion of enterovirus. [2C] Control samples (unnatural traffic accident victims) showing no significant pathological findings.

Figure 3

Immunohistochemical studies for the identification of infected immune cells in the myocardium of sudden unexpected death victims. [3A] Immunohistochemical labeling of paraffin-embedded tissue sections with antibody recognizing T cell (arrows). [3B] Immunohistochemical labeling of paraffin-embedded tissue sections with antibody recognizing B cell (arrows).

Figure 4

Detection of Coxsackie B enterovirus RNA by RT-PCR in post-mortem myocardial samples from Sudden Unexpected Death victims (1 and 10: molecular size marker 100-bp DNA ladder; 2: Negative control RNA extraction; 3: Negative control RT-PCR mixture; 4 to 6 and 8: Samples from coxsackie B enterovirus-positive cases; 7: Samples from a coxsackie B enterovirus-negative case; 9: A positive control; coxsackievirus B3: 155 bp).