Coxsackie B3 myocarditis in 4 cases of suspected sudden infant death syndrome: diagnosis by immunohistochemical and molecular-pathologic investigations

Immunohistochemical and molecular-pathologic techniques have improved the diagnosis of myocarditis as compared with conventional histologic staining methods done according to the Dallas criteria. Most investigations were carried out on adults, and only a few authors investigating childhood deaths applied these modern methods, used for diagnosing myocarditis. We report on four children under one year of age, who suddenly died without prodromal symptoms. Their deaths were attributed to SIDS (sudden infant death syndrome). Immunohistochemical (LCA, CD68, CD45R0, MHC-class-II-molecules, VP1-capsid-protein of enteroviruses) and molecular-pathologic (RT-PCR) investigations, however, suggested that death was caused by a coxsackie-B3-myocarditis. In the future, these methods should be used for investigating cases with suspicion of SIDS.     

Molecular pathology of inflammatory cardiomyopathy

Endomyocardial biopsy (EMB) is often performed in patients presenting with sudden onset of heart failure to identify myocarditis. The introduction of immunohistochemical techniques for the detection and differentiation of infiltrating immune cells, specific adhesion molecules and MHC class I and II molecules increased the prognostic value of EMB in the diagnosis of myocarditis considerably. A major breakthrough in the understanding of pathogenetic mechanisms in myocarditis was achieved by diagnostic use of molecular biological methods. By application of in situ hybridization and PCR, enteroviruses, and more recently, parvovirus B19 (PVB19) have been identified as relevant agents of myocarditis. The different cell tropism of these viruses implicates distinct pathogenic principles, which, at present, are not completely understood. Whereas enteroviruses damage the heart primarily via direct lysis of infected myocytes, PVB19 does not infect myocytes, but endothelial cells of small intracardiac arterioles and venules, resulting in impairment of myocardial microcirculation with secondary myocyte necrosis during acute infection. Histological and immunohistological stainings combined with molecular biological approaches in EMB will help us to resolve the question of whether patients with myocarditis should be treated by specific antiviral agents or by immunosuppressive therapies.     

Parvovirus B19 is a bystander in adult myocarditis

BackgroundThe genomic DNA of parvovirus B19, a small single-stranded DNA virus of the genus Erythrovirus, has been shown to persist in solid tissues of constitutionally healthy, immunocompetent individuals. Despite these data, many case reports and series have linked the presence of parvovirus B19 genomic DNA, detected through nucleic acid amplification testing, with myocarditis and cardiomyopathy. Herein, we use multiple tools to better assess the relationship between parvovirus B19 and myocarditis and cardiomyopathy.MethodsNucleic acid amplification testing, immunohistochemistry, in situ hybridization, and electron microscopy were used to assess the location and activity of parvovirus B19 in cases of myocarditis and in cases with no significant cardiac disease.ResultsNucleic acid amplification testing for parvovirus B19 genomic DNA was positive in 73% of patients with myocarditis/cardiomyopathy and in 26% of patients with no significant disease. In situ hybridization and immunohistochemistry showed that, in cases with amplifiable parvovirus B19 DNA, parvovirus B19 genomic DNA and viral protein production were present in rare mononuclear cells.ConclusionsIn a majority of cases of myocarditis and a significant number of otherwise normal hearts, nucleic acid amplification testing detected persistent parvovirus B19 genomic DNA that did not play a significant pathogenic role. The source of parvovirus B19 DNA appeared to be interstitial mononuclear inflammatory cells and not myocardial or endothelial cells. Therefore, nucleic acid amplification testing alone is not diagnostically helpful for determining the etiology of adult myocarditis.     

心源性猝死心肌细胞凋亡与c-jun基因蛋白的表达

目的 探讨心肌细胞凋亡和c-jun基因蛋白在心源性猝死(SCD)中的表达及其法医学意义. 方法 50例法医尸检案例,其中冠心病猝死组16例,病毒性心肌炎猝死组14例,扩张型心肌病猝死组10例及对照组(非SCD猝死病例)10例.通过TUNEL法和免疫组织化学法对各组心肌细胞凋亡指数、c-jan基因蛋白表达情况的吸光度进行半定量检测,并分析各组之间的差异. 结果 冠心病猝死组、病毒性心肌炎猝死组、扩张型心肌病猝死组的凋亡指数均显著高于对照组(P<0.01).病毒性心肌炎猝死组和扩张型心肌病猝死组的凋亡指数均高于冠心病猝死组(P<0.01),前两者之间差异无统计学意义(P>0.05).冠心病猝死组、病毒性心肌炎猝死组、扩张型心肌病猝死组的c-jan基因蛋白吸光度值均显著高于对照组(P<0.01).3组之间的吸光度值差异无统计学意义(P>0.05). 结论 应用TUNEL法检测心肌细胞凋亡和免疫组织化学法检测心肌细胞内c-jun基因蛋白的表达,可作为法医鉴定SCD的重要指标.     

Fatal parvovirus myocarditis in a 5-year-old girl

Infection with parvovirus B19 is common in children and typically causes mild illness. We report here the case of a 5-year-old girl who died suddenly, 2 weeks after the clinical diagnosis of a parvoviral infection (erythema infectiosum). Microscopic examination of the heart showed severe myocarditis with extensive T-cell and macrophage infiltration. Cultures, serology, and molecular analyses of serum for enteroviridae, adenovirus, influenza, varicella zoster, cytomegalovirus, and herpes simplex viruses were negative. Quantitative polymerase chain reaction (PCR) analysis for parvovirus B19 in peripheral blood, however, showed active infection (91,000 genomes/mL serum; 2.4 genomes/mononuclear cell). Despite the presence of myocarditis, immunostaining for parvoviral surface antigens was negative in the heart. Quantitative PCR analysis of paraffin sections showed that myocardial parvoviral content was significantly less than that of the normal appearing kidney and within the range predicted simply by tissue blood content. Thus, parvovirus B19 infection can be complicated by fatal myocarditis. Because the virus does not appear to have infected the heart, per se, we speculate that myocarditis arose from immunological cross-reaction to epitopes shared between the virus and the myocardium. HUM PATHOL 32:342-345.     

Sudden infant death syndrome associated with rotavirus infection

Rotavirus was detected in the stools of five children stricken with sudden infant death syndrome (SIDS) over a three-week period. While none of the children had acute gastroenteritis, four of the five had acute upper respiratory infections. Rotavirus was also identified in tracheal aspirates from two of the infants. Extensive investigations failed to reveal the presence of any other viruses or toxins in specimens obtained from the five children with SIDS. Rotavirus was not found in the stool specimens obtained from a control group of 36 infants including six who died of causes other than SIDS. Future attempts at the prevention of rotavirus infections should be directed at populations susceptible to sudden infant death syndrome.     

Fulminant parvovirus B19‐associated pancarditis with haemophagocytic lympho‐histiocytosis in an immunocompetent adult

Myocarditis is a common cardiac disease that is identified on routine postmortem examinations. Initially, coxsackie viruses, other enteroviruses and adenoviruses were thought to be more common causes of myocarditis; however, recently, parvovirus B19 (PVB19) as well as human herpesvirus 6 (HHV6) have entered the arena. We describe autopsy findings of a patient who had a lethal myocarditis with haemophagocytic lympho‐histiocytosis in a course of systemic PVB19 infection. The present case illustrates the unusually severe and rapid course of PVB19 myocarditis with associated haemophagocytic lympho‐histiocytosis leading to death.     

Apparent hypoxic changes in pulmonary arterioles and small arteries in infancy.

The pulmonary arterioles and small arteries were studied and their musculature and its nuclei were quantified in 90 neonates, infants, and young children who had suffered from a variety of clinical and hypoxic conditions immediately before death. Among the 90 cases investigated in this study, 30 were of sudden infant death syndrome (SIDS). No evidence was found to support the view that cases of SIDS are subjected to chronic hypoxia before death as significantly more medial muscle tissue in the pulmonary arterioles and small arteries was found in the chronic hypoxic group compared to the SIDS, non-hypoxic, and acute hypoxic groups. Furthermore, there was no statistically significant difference in the amount of medial muscle tissue of the pulmonary vessels as between the SIDS, non-hypoxic, and acute hypoxic groups. With other signs of acute hypoxia found at the necropsy of SIDS, the results of this study could be considered to support the view that cases of SIDS succumb as a result of an acute episode of hypoxia, or possibly repeated short-duration episodes of acute hypoxia which do not produce pulmonary vascular changes.     

Prevalence of human parvovirus B19 DNA in cardiac tissues of patients with congenital heart diseases indicated by nested PCR and in situ hybridization.

BACKGROUND: Infection with parvovirus B19 (B19) was reported to be correlated with myocarditis, cardiomyopathy, and kawasaki disease. But no information is available about the relationship between inutero B19 infection and congenital heart disease (CHD). OBJECTIVE: To explore whether there is relationship between B19 infection and CHD. STUDY DESIGN: Retrospective investigation of biopsy samples from CHD patients from January 1996 to December 1998. METHODS: Parvovirus B19 was detected in biopsy samples from 42 cases of CHD patients and 38 cases of biopsy or autopsy samples from patients with other diseases (controls) by nested polymerase chain reaction (PCR) and in situ hybridization (ISH) technique. HE staining was also performed to observe the morphology of these cardiac tissue samples. RESULTS: Nested PCR assay indicated that seven of 42 (16.7%) CHD cardiac tissue were B19 DNA positive, while all the 38 controls were B19 DNA negative, the difference is significant (P = 0.012). ISH assay indicated that five of 42 (11.7%) CHD cardiac tissues were positive for B19 DNA and none of the control cardiac tissue were positive, all B19 DNA positive signals were located in the nucleuses of cardiac cells. HE staining showed that there was no inflammatory change in B19 DNA positive cardiac tissue. CONCLUSIONS: Parvovirus B19 DNA was presented in part of CHD cardiac tissues and located in nucleus, which suggested that inutero B19 infection might be correlated with CHD.     

Coincidence of different structures of mucosa-associated lymphoid tissue (MALT) in the respiratory tract of children: no indications for enhanced mucosal immunostimulation in sudden infant death syndrome (SIDS)

Mucosa-associated lymphoid tissue (MALT) is the principal inductive site for mucosal immune responses that are capable of T and B cell responses and antigen-specific responses. In previous independent studies different structures of MALT, e.g. bronchus-, larynx- and nose-associated lymphoid tissue (BALT, LALT, NALT) have been described separately in various frequencies in the human respiratory tract over life spans. Because upper respiratory tract infections are common in infants, dysregulations of mucosal immune responses might be seriously involved in the aetiology of sudden infant death syndrome (SIDS). In the present study the coincidental occurrence of the three different MALT structures in the respiratory tract within the same patients were studied, and cases of SIDS and children who had died from different traumatic and natural causes of death (non-SIDS) were compared. First, the frequency of BALT and LALT in 46 children (35 SIDS, 11 non-SIDS) with or without NALT were examined. A tendency was found of a coincidence of respiratory MALT structures. In 50 additional cases of infant death (30 SIDS, 20 non-SIDS) from the multi-centric German Study on Sudden Infant Death Syndrome (GeSID) where death had occurred in the first year of life, the coincidence was evaluated. A coincidental occurrence of BALT, LALT and NALT or BALT and LALT (each about 30%) was found in both groups, whereby the coincidence in SIDS and the control patients did not differ. Interestingly, the children with coincidental MALT were strikingly older, supporting the hypothesis of respiratory MALT formation via environmental stimulation over time.     

The cardiac conduction system in sudden infant death syndrome: a report on three cases.

The cardiac conduction systems in three cases of sudden infant death syndrome (SIDS) were compared with those in children dying from known causes. Foci of inflammatory cells and areas of necrosis were found in SIDS. Lymphocytes and eosinophils were counted in the adjacent myocardium and significantly larger numbers were present in SIDS. These parhological changes in the conduction systems may be capable of producing cardiac arrhythmia and sudden death.     

Histologic and in situ viral findings in the myocardium in cases of sudden, unexpected death.

The purpose of this study was to do in situ viral detection in myocardial tissues of individuals who suffered sudden unexpected death and to correlate the results with the postmortem histopathologic findings. Thirteen cases were identified and the heart tissues were analyzed for adenovirus, cytomegalovirus, Epstein Barr virus, herpes simplex virus 1 and 2, human immunodeficiency virus 1 (HIV-1), influenza A, influenza B, parvovirus, rotavirus, picornavirus (including separate primers for enterovirus and Coxsackie virus A and B), varicella zoster virus, and respiratory syncytial virus. Thirteen individuals aged 2 to 67 years were studied. In each case, polymerase chain reaction-amplified viral RNA was detected in situ: Coxsackie virus B (5 cases), rotavirus (4 cases), HIV-1 (2 cases), influenza A (1 case), and influenza B (1 case). Immunohistochemical detection of viral proteins was found in the five Coxsackie virus cases and four rotavirus cases. The mononuclear inflammatory infiltrate was diffuse and marked only in the cases of influenza A and HIV-1, as well as one of the Coxsackie virus and rotavirus cases, respectively. Immunohistochemical analysis showed that the most common cell type in the inflammatory infiltrates was CD68-positive macrophages. Direct myocyte infection was most prominent in the cases of Coxsackie virus infection. In summary, in situ viral detection was documented in each case of idiopathic myocarditis associated with sudden, unexpected death; in 6/13 cases, the myocarditis was focal and minimal. Although Coxsackie virus was, as expected, the most common virus noted, other viruses including rotavirus and HIV-1 were also observed, highlighting the need for comprehensive viral and histologic analyses in such cases.     

Fatty acid oxidation disorders as primary cause of sudden and unexpected death in infants and young children: an investigation performed on cultured fibroblasts from 79 children who died aged between 0-4 years.

BACKGROUND: Disorders of fatty acid metabolism are known to be responsible for cases of sudden and unexpected death in infancy. At least 14 disorders are known at present. 120 cases of sudden infant death syndrome (SIDS) had been examined for a prevalent mutation (G985) causing medium chain acyl CoA dehydrogenase deficiency, which is inherited in an autosomal recessive mode. No over-representation of either homozygous or heterozygous cases was found. AIMS: To investigate a broader spectrum of fatty acid oxidation disorders in a wider range of sudden deaths in infants and young children. METHODS: Seventy nine cases of unexpected death in infants and young children younger than 4 years old were examined for a minimum of nine fatty acid oxidation disorders, using the global [9, 10-3H] myristic acid oxidation assay in cultured fibroblasts from achilles tendon biopsies taken at postmortem examination. RESULTS: Three cases with fatty acid oxidation disorders and two carriers of the G985 mutation were found, all categorized as non-SIDS or borderline SIDS. The global assay used has the advantage of simplicity. CONCLUSIONS: These results indicate that disorders of fatty acid oxidation play a small but significant role in the cause of unexpected death in infants and young children, and that infants and children dying in this way should be regarded as high risk candidates for metabolic diseases.     

Pancreatic islet abnormalities in sudden infant death syndrome. Qualitative and quantitative analyses of 15 cases.

The pancreata of 15 autopsy cases of sudden infant death syndrome (SIDS) and those of 14 age-matched controls were examined qualitatively and quantitatively to re-evaluate the relationship between pancreatic islet abnormalities and sudden death in infancy. Histopathologically, a diffuse or focal form of nesidioblastosis and septal islets were frequently observed in the pancreata of both groups. Endocrine cell dysplasia was found only in 2 infants who had died of SIDS. Quantitatively, there was little difference of islet cell composition between the SIDS cases and the controls. A relatively high proportion of islet cell area to total pancreatic tissue area was demonstrated in the SIDS group (8.46 +/- 4.90% in the pancreatic head; 8.66 +/- 4.23% in the pancreatic body to tail) in comparison with the controls (5.32 +/- 1.77%; 5.63 +/- 1.60%). Although nesidioblastosis and septal islets were considered to be within the limits of normal variation during pancreatic development, endocrine cell dysplasia and quantitatively unusual proliferation of the pancreatic endocrine tissue suggest the possibility that abnormalities in the endocrine pancreas may be causally related to sudden death in infancy.     

Detection and significance of adenoviruses in cases of sudden infant death

Respiratory tract infections have been thought to act as a trigger mechanism in sudden infant death. In 118 autopsy cases of infant death, paraffin-embedded or frozen lung tissues were investigated by means of a nested polymerase chain reaction (PCR) to detect adenovirus (AV) DNA. The primers used are general primers and allow the detection of most pathogenic adenoviruses with high specificity and sensitivity and independently of devitalization of viruses or degradation of viral DNA. For the investigation three groups were established: there were 13 cases of unnatural death, 78 cases of natural death without histological signs of interstitial pneumonia, and 27 cases with interstitial pneumonia. The first group was AV negative. In the group without interstitial pneumonia AV was detected in 10.2% of the cases. In the group with interstitial pneumonia the frequency of AV detection was almost 26%. The results obtained demonstrate an association between interstitial pneumonia and detection of AV DNA, indicating that AV may play an important part in pulmonary infection in infants. Histological evidence of interstitial pneumonia was not observed in all AV-positive cases, perhaps because nonspecific virus-related changes occurred only in early stages of viral infection. Comparison of the AV frequency in SIDS (15%) and non-SIDS cases (4%) indicates an association between pulmonary AV infections and sudden death. These results support the working hypothesis of respiratory infections acting as a trigger mechanism in sudden infant death.     

Childhood myocarditis and parvovirus B19 genotypes

BackgroundHuman parvovirus B19 (PVB19) infection is occasionally associated with acute myocarditis. Three cases of children with PVB19 virus-associated myocarditis occurred in a very short period and the same geographical region.ObjectiveTo elucidate if virological factors could be responsible for determining the course of infection, a molecular epidemiologic investigation was performed.Study DesignThe diagnosis of myocarditis was established by histology or echocardiography. In the three cases, the PVB19 DNA was detected in different samples. Eight different regions were amplified by PCR using a high fidelity Taq polymerase and sequenced on both strands. Phylogenetic analyses were performed. First, the genotypes of the PVB19 strains were determined, then the intra-patient viral variability was analysed by sequencing PVB19 detected in different specimens sampled from the same patient at the same moment.ResultsNearly complete sequences of the PVB19 virus (4265nt) were obtained from different samples in the three patients. The phylogenetic analyses showed that PVB19 strains identified clustered with genotype 1a PVB19 strains referenced in GenBank. When compared to the referenced strain NC_000883, the number of substitutions (transitions and transversions) were as follows: 58 for Caen.FRA/19.09, 74 for Caen.FRA/21.09 and 60 for Caen.FRA/24.09. The strains isolated from the same patient showed 100% of similarity.ConclusionsViral myocarditis is a frequently unrecognized cause of post-inflammatory cardiomyopathy. The detailed molecular analyses do not give rise to virological markers associated with myocarditis in these children.     

Fibromuscular hyperplasia of the pulmonary artery in sudden infant and perinatal unexpected death

IntroductionThe purpose of this study was to describe cases presenting with fibromuscular hyperplasia of the pulmonary arteries that could belong to the group of sudden infant death syndrome (SIDS) and sudden unexpected perinatal death “gray zone” or borderline cases.MethodsIn a total of 12 cases, eight females and four males, ranging in age from 39 gestational weeks to 93 postnatal days, dying suddenly and unexpectedly, a fibromuscular hyperplasia of the pulmonary artery was detected. Postmortem examinations were requested with a clinical SIDS or sudden unexpected perinatal death. A complete autopsy was performed, including close examination of the brainstem and cardiac conduction system.ResultsHistological examination showed the presence of various degrees of fibromuscular hyperplasia with fibrosis of the right (six cases), left (five cases) or both (one case) pulmonary arteries.ConclusionsIn our cases, fibromuscular hyperplasia of the pulmonary artery alone might or might not have accounted for the sudden deaths, if it had not been for the concomitant presence of hypoplasia of the arcuate nucleus in the brainstem and/or cardiac conduction system abnormalities. Therefore, they were classified as SIDS/sudden unexpected perinatal death gray zone or borderline cases. Necropsy studies of sudden infant and perinatal death should always include an accurate gross and histological examination of the pulmonary arteries, as well as of the brainstem and cardiac conduction system.