Debra L. Kearney

Detection of viruses in myocardial tissues by polymerase chain reaction : evidence of adenovirus as a common cause of myocarditis in children and adults

OBJECTIVES The purpose of this study was to analyze cardiac tissue and blood for viral genomes using polymerase chain reaction (PCR) to define the common viral etiologies of myocarditis by age group. BACKGROUND Enteroviruses are considered the most common cause of myocarditis at all ages. Diagnosis relies on viral cultures, serology, and cardiac histology, which lack sensitivity, as well as PCR. However, in many cases enteroviruses are not detected. METHODS Cardiac samples were obtained for PCR analysis from patients with myocarditis (n = 624) and dilated cardiomyopathy (DCM) (n = 149). Patients were analyzed by age group, including neonates (n = 116), infants (n = 191), toddlers (n = 87), children (n = 110), adolescents (n = 92), and adults (n = 177). After nucleic acids had been extracted from an endomyocardial biopsy, an explant, or autopsy samples, PCR and reverse transcription PCR were performed to detect the genomic sequences of enterovirus, adenovirus, cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), parvovirus, respiratory syncytial virus (RSV), and influenza A virus. RESULTS Viral genome was amplified (adenovirus = 142, enterovirus = 85, CMV = 18, parvovirus = 6, influenza A = 5, HSV = 5, EBV = 3, RSV = 1) from 239 (38%) of the 624 samples from myocarditis patients, including 26 patient samples in which dual infection was found. Virus was detected in 30 (20%) of 149 DCM patient samples; only adenovirus (n = 18) and enterovirus (n = 12) were detected. CONCLUSIONS Polymerase chain reaction identified adenovirus as the most common virus in the myocardium of children and adults with myocarditis and DCM. Although enteroviruses are also found in these patients, they appear to be a less common cause of myocarditis than adenovirus.

Acute myocarditis. Rapid diagnosis by PCR in children.

The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histological criteria. Viral cultures and serology are often unrewarding, with low yields. The purpose of this study was to analyze the usefulness of polymerase chain reaction (PCR) in the rapid diagnosis of acute myocarditis in children. Methods and ResultsPCR was used to analyze 38 myocardial tissue samples from 34 patients with suspected acute viral myocarditis and 17 control patients with congenital heart disease (14) or hypertrophic cardiomyopathy (3). Myocardial samples were obtained at the time of right ventricular biopsy (13 samples), from explanted hearts (18 samples) at transplantation, and from cardiac autopsy specimens (24 samples) and were evaluated for the presence of enterovirus, cytomegalovirus (CMV), adenovirus, and herpes simplex virus (HSV) using PCR primers designed to consensus and unique sequences of these viral genomes. Blood also was obtained at the time of biopsy (11) or transplant (18). In 26 of 38 myocardial samples (68%), viral genome was detected by PCR (15 adenoviral, 8 enteroviral, 2 HSV, 1 CMV), whereas all control myocardial samples and blood samples were negative. Four patients had positive viral cultures, and these matched the PCR findings. Disagreement with histopathology occurred in 13 of 26 PCRpositive specimens, usually associated with adenovirus. ConclusionsPCR offers a rapid, sensitive diagnostic method for myocardial viral infection. While enterovirus is an important etiological agent, adenovirus was more prevalent in this series and should be evaluated when etiology is sought. PCR used in conjunction with standard endomyocardial biopsy appears to enhance the likelihood of detecting viral genome in the myocardium of patients with clinical evidence of myocarditis.

Evaluation of Postmortem Endomyocardial Biopsy Specimens From 38 Patients With Lymphocytic Myocarditis: Implications for Role of Sampling Error

Among 38 hearts from autopsies in which lymphocytic myocarditis contributed to death, 10 endomyocardial specimens from the apical septal aspect of each ventricle (760 specimens) and 6 slices of ventricular myocardium (228 slices) were evaluated for myocarditis by the Dallas criteria. For each case, the number of positive biopsy samples correlated well with the mean lymphocyte counts in biopsy tissues (P less than 0.0001) and the mean number of inflammatory foci per square centimeter in myocardial slices (P less than 0.001). Right ventricular biopsy specimens, however, were positive in only 63% of the 38 cases and 17% of the 380 specimens. Similarly, left ventricular biopsy tissues were positive in only 55% of the cases and 20% of the specimens. Sampling error was somewhat more prevalent among the 11 cases with isolated myocarditis than in the 27 with myocarditis and other illnesses. Even when 10 biopsy specimens per ventricle were evaluated, the frequency of false-negative results was 45% for the left and 37% for the right ventricle. Although myocarditis was noted in 68% of the 38 septal slices, it involved the subendocardium of the right ventricle (from which biopsy specimens are usually obtained) in only 24%. Because of the mild and focal nature of the inflammatory infiltrates and involvement of regions inaccessible to the bioptome, sampling error contributes appreciably to false-negative results in endomyocardial biopsy tissue from patients with myocarditis. Thus, when myocarditis is evaluated by biopsy alone, only positive findings are considered diagnostic.

Desmosomal Dysfunction due to Mutations in Desmoplakin Causes Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

Implantation of balloon-expandable intravascular grafts by catheterization in pulmonary arteries and systemic veins.

The purpose of this investigation was to evaluate the efficacy and safety of implanting expandable intravascular stents in pulmonary arteries and systemic veins. Twenty-seven balloon-expandable grafts were placed in 13 mongrel dogs under anesthesia. A long sheath was introduced over a wire and catheter or dilator into the pulmonary artery or target vein. A collapsed stainless steel expandable mesh stent was placed over the balloon of an angioplasty catheter. The catheter with the mounted stent was advanced through the sheath. The stent expanded to the diameter of the balloon as the balloon was inflated, and remained expanded as the balloon was deflated. The stent was expanded further with a larger balloon in 11 instances. Eleven stents were placed successfully in pulmonary arteries (out of thirteen attempted), and 11 of 14 were installed in tributaries of the precava or postcava. Three inadvertent embolizations of the devices occurred. All three devices that embolized lodged in the pulmonary arteries and did not obstruct flow. Seven dogs were recatheterized at intervals ranging from 56 to 278 days. Twelve stents were patent and nonobstructive, and two were malpositioned, one of which was obstructed. Three animals were killed 2 months (two dogs) and 9 months (one dog) after the implantations. The stents (four in the pulmonary arteries and two in veins) were completely covered with neointima and were patent, without thrombosis. These stents hold promise for definitive dilation of congenital or postoperative vessel stenoses.

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Association of Parvovirus B19 Genome in Children With Myocarditis and Cardiac Allograft Rejection Diagnosis Using the Polymerase Chain Reaction

BACKGROUND Inflammatory diseases of the heart, including myocarditis and cardiac transplant rejection, are important causes of morbidity and mortality in children. Although viral infection may be suspected in either of these clinical conditions, the definitive etiology is often difficult to ascertain. Furthermore, the histology is identical for both disorders. Coxsackievirus has long been considered the most common cause of viral myocarditis; however, we previously demonstrated by polymerase chain reaction (PCR) analysis that many different, and sometimes unexpected, viruses may be responsible for myocarditis and cardiac rejection. In this study, we describe the association of parvovirus genome identified through PCR analysis of cardiac tissue in the clinical setting of myocarditis and cardiac allograft rejection. METHODS AND RESULTS Myocardial tissue from endomyocardial biopsy, explant, or autopsy was analyzed for parvovirus B19 using primers designed to amplify a 699-base pair PCR product from the VP1 gene region. Samples tested included those obtained from patients with suspected myocarditis (n=360) or transplant rejection (n=200) or control subjects (n=250). Parvoviral genome was identified through PCR in 9 patients (3 myocarditis; 6 transplant) and no control patients. Of the 3 patients with myocarditis, 1 presented with cardiac arrest leading to death, 1 developed dilated cardiomyopathy, and the other gradually improved. Four of the 6 transplant patients had evidence of significant rejection on the basis of endomyocardial biopsy histology. All transplant patients survived the infection. CONCLUSIONS Parvovirus is associated with myocarditis in a small percentage of children and may be a potential contributor to cardiac transplant rejection. PCR may provide a rapid and sensitive method of diagnosis.

Sudden Death and Cardiovascular Collapse in Children With Restrictive Cardiomyopathy

BACKGROUND Restrictive cardiomyopathy (RCM) is rare in children, and the prognosis is poor. In the present study, we evaluated all pediatric patients with RCM who were at our institution during a 31-year period to determine the clinical outcome and cause of death. Those who sustained sudden, unanticipated cardiac arrests were evaluated for risk factors that are predictive of sudden death. METHODS AND RESULTS Eighteen consecutive patients were reviewed. Presentation, clinical course, laboratory data, and histopathological evidence of ischemia were compared between patients with and without sudden death events. The results demonstrated that patients who were at risk for sudden death were girls with chest pain, syncope, or both at presentation and without congestive heart failure. Although not statistically significant for sudden death, Holter monitor evidence of ischemia predicted death within months. Histopathological evidence of acute or chronic ischemia was found in the majority of patients, with acute ischemia more common among those who sustained sudden death events. CONCLUSIONS All children with RCM are at risk for ischemia-related complications and death, and some are at risk of sudden death. In the present study, patients at risk of sudden death appeared well and had no evidence of ongoing heart failure but often had signs or symptoms of ischemia characterized by chest pain, syncope, or both. ECGs and Holter monitors may be useful screening tools. The use of beta-blockade, the placement of an implantable cardioverter-defibrillator, and preferential status 1A or B listing for cardiac transplantation are proposed for pediatric patients with RCM and evidence of ongoing ischemia.

Danon disease as an underrecognized cause of hypertrophic cardiomyopathy in children

Background—Some patients with hypertrophic cardiomyopathy (HCM) or left ventricular hypertrophy also present with skeletal myopathy and Wolff-Parkinson-White (WPW) syndrome; mutations in the gene encoding the lysosome-associated protein-2 (LAMP-2) have been identified in these patients, suggesting that some of these patients have Danon disease. In this study we investigated the frequency of LAMP2 mutations in an unselected pediatric HCM population. Methods and Results—LAMP2 was amplified from genomic DNA isolated from peripheral lymphocytes of 50 patients diagnosed with HCM and analyzed by direct DNA sequencing. In 2 of the 50 probands (4%), nonsense mutations were identified. In 1 family the proband initially presented with HCM as a teenager, which progressed to dilated cardiomyopathy (DCM) and heart failure. Skeletal myopathy and WPW were also noted. The teenage sister of the proband is a carrier of the same LAMP2 mutation and has HCM without skeletal myopathy or WPW. The other proband presented with HCM, WPW, and skeletal myopathy as a teenager, whereas his carrier mother developed DCM during her 40s. Skeletal and cardiac muscle sections revealed the absence of LAMP-2 on immunohistochemical staining. Conclusions—LAMP2 mutations may account for a significant proportion of cases of HCM in children, especially when skeletal myopathy and/or WPW is present, suggesting that Danon disease is an underrecognized entity in the pediatric cardiology community.

Incessant ventricular tachycardia in infants: myocardial hamartomas and surgical cure.

Infants with incessant ventricular tachycardia (occurring greater than 10% of the day) have generally been described in pathologic studies. This report describes 21 patients with incessant ventricular tachycardia present greater than 90% of the day and night; the age at diagnosis ranged from birth to 30 months (mean 10.5 months). The most common clinical presentation was cardiac arrest (11 patients, in 5 after digitalis for presumed supraventricular tachycardia); another 6 patients had congestive heart failure and 4 were asymptomatic. Three patients had coexisting Wolff-Parkinson-White syndrome. The rate of incessant ventricular tachycardia ranged from 167 to 440 (mean 260 beats/min) and the QRS duration from 0.06 to 0.11 second. The most common electrocardiographic (ECG) pattern (10 of 21) was right bundle branch block with left axis deviation, but other right and left bundle branch block patterns were observed. Conventional and investigational antiarrhythmic agents (nine patients received amiodarone) failed to eliminate incessant ventricular tachycardia in all. Electrophysiologic studies localized incessant ventricular tachycardia to the left ventricle in 17 (to the apex in 2, the free wall in 9 and the septum in 6) and to the right ventricular septum in 4. No structural abnormalities were found on the echocardiogram or angiocardiogram. All 21 patients had surgery at an age of 3.5 to 31 months (mean 16). In 15 a tumor was found: 13 myocardial hamartomas (9 discrete, 4 diffuse throughout both ventricles) and 2 rhabdomyomas (1 multiple). Myocarditis was found in one patient (the oldest). In four, only myocardial fibrosis was found; results of one biopsy were normal.(ABSTRACT TRUNCATED AT 250 WORDS)