Jiyuan Ni

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.

Association of Viral Genome with Graft Loss in Children after Cardiac Transplantation

BACKGROUND The survival of recipients of cardiac allografts is limited by rejection, lymphoproliferative disease, and coronary vasculopathy. The purpose of this study in children who had received heart transplants was to evaluate the cardiac allografts for myocardial viral infections and to determine whether the presence of viral genome in the myocardium correlates with rejection, coronary vasculopathy, or graft loss. METHODS We enrolled heart-transplant recipients 1 day to 18 years old who were undergoing evaluation for possible rejection and coronary vasculopathy. Endomyocardial-biopsy specimens were evaluated for evidence of rejection with the use of standard criteria and were analyzed for the presence of virus by the polymerase chain reaction (PCR). RESULTS PCR analyses were performed on 553 consecutive biopsy samples from 149 transplant recipients. Viral genome was amplified from 48 samples (8.7 percent) from 34 patients (23 percent); adenovirus was found in 30 samples, enterovirus in 9 samples, parvovirus in 5 samples, cytomegalovirus in 2 samples, herpes simplex virus in 1 sample, and Epstein-Barr virus in 1 sample. In 29 of the 34 patients with positive results on PCR (85 percent), an adverse cardiac event occurred within three months after the positive biopsy, and 9 of the 34 patients had graft loss due to coronary vasculopathy, chronic graft failure, or acute rejection. In 39 of the 115 patients with negative results on PCR (34 percent), an adverse cardiac event occurred within three months of the negative PCR finding; graft loss did not occur in any of the patients in this group. The odds of graft loss were 6.5 times as great among those with positive results on PCR (P=0.006). The detection of adenovirus was associated with considerably reduced graft survival (P=0.002). CONCLUSIONS Identification of viral genome, particularly adenovirus, in the myocardium of pediatric transplant recipients is predictive of adverse clinical events, including coronary vasculopathy and graft loss.

The detection of cardiotropic viruses in the myocardium of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

OBJECTIVES We sought to investigate the role of cardiotropic viruses, including adenovirus, cytomegalovirus (CMV), enterovirus and parvovirus, in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). BACKGROUND Arrhythmogenic right ventricular dysplasia/cardiomyopathy is characterized by a gradual loss of myocytes, inflammatory infiltrates and replacement by fatty and fibrous tissue. It has been speculated that ARVD/C is a sequela of viral myocarditis in some patients, and the role of the coxsackievirus B3 has been debated. METHODS Myocardial samples from 12 patients with ARVD/C were analyzed by polymerase chain reaction for the presence of cardiotropic viruses. RESULTS Enteroviral sequences were detected in seven patients and adenovirus type 5 in another two patients. During the same period, 215 control samples were analyzed in which only CMV (n = 2) and enterovirus (n = 1) were detected. This suggests a link between ARVD/C and the presence of viral genome (enterovirus or adenovirus) in the myocardium. CONCLUSIONS We report that cardiotropic viruses are more frequently identified in patients with ARVD/C than in control subjects. However, the role of these viruses in ARVD/C pathogenesis remains unknown.

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.

Viral Infection of the Myocardium in Endocardial Fibroelastosis Molecular Evidence for the Role of Mumps Virus as an Etiologic Agent

BACKGROUND Endocardial fibroelastosis, previously a common disease of children, often resulted in congestive heart failure and death. Virus-induced myocarditis was the suspected first step in the pathogenesis of the disease, with enteroviruses and mumps virus considered potential causes. Direct evidence for their involvement was limited, however, and during the past two decades, a significant decline in the incidence of endocardial fibroelastosis occurred. Recently, we demonstrated polymerase chain reaction to be a rapid and sensitive method for identification of the viral genome in the myocardium of patients with myocarditis and dilated cardiomyopathy. The purpose of this study was to analyze myocardial samples of patients with endocardial fibroelastosis for the viral genome. METHODS AND RESULTS Myocardial samples from 29 patients with autopsy-proven endocardial fibroelastosis were analyzed for viral genome (enterovirus, adenovirus, mumps, cytomegalovirus, parvovirus, influenza, herpes simplex virus) by use of polymerase chain reaction or reverse transcriptase-polymerase chain reaction. In 90% of samples, the viral genome was amplified; > 70% of the samples were positive for mumps viral RNA, while 28% amplified adenovirus. In contrast, only 1 of 65 control samples amplified a virus (enterovirus). Two regions of mumps virus were amplified: the nucleocapsid gene and the polymerase-associated protein gene. Interestingly, only 3 of the 21 samples that were positive for mumps RNA were positive with both sets of primers, indicating that the persistence of mumps virus in the myocardium may be related to the selection of defective virus mutants. CONCLUSIONS These data suggest an etiologic role for viral infection in endocardial fibroelastosis, supporting the hypothesis that endocardial fibroelastosis is a sequela of a viral myocarditis, in particular of that due to mumps virus.

The detection of viral genomes by polymerase chain reaction in the myocardium of pediatric patients with advanced HIV disease

OBJECTIVES The aim of this study was to investigate the frequency of viral nucleic acid detection in the myocardium of human immunodeficiency virus (HIV)-infected children to determine whether an association exists with the development of heart disease. BACKGROUND As improved medical interventions increase the life expectancy of HIV-infected patients, increased incidences of myocarditis and dilated cardiomyopathy (DCM) are becoming more apparent, even in patients without clinical symptoms. METHODS Myocardial samples were obtained from the postmortem hearts of 32 HIV-infected children and from 32 age-matched controls consisting of patients with structural congenital heart disease and no myocardial inflammation and no cardiac or systemic viral infection. The hearts were examined histologically and analyzed for the presence of viral sequences by polymerase chain reaction (PCR) or reverse transcription-PCR. RESULTS Myocarditis was detected histologically in 11 of the 32 HIV-infected patients, and borderline myocarditis was diagnosed in another 13 cases. Infiltrates were confined to the epicardium in two additional hearts. Virus sequences were detected by PCR in 11 of these 26 cases (42.3%); adenovirus in 6, CMV in 3 and both adenovirus and CMV in 2. Two cases without infiltrates were also positive for adenovirus: one had congestive heart failure (CHF) and the other adenoviral pneumonia. No other viruses were detected by PCR, including HIV proviral DNA. All control samples were negative for all viruses tested. CONCLUSIONS These data suggest that the presence of viral nucleic acid in the myocardium is common in HIV-infected children, and may relate to the development of myocarditis, DCM or CHF and may contribute to the rapid progression of HIV disease.

Tracheal Aspirate as a Substrate for Polymerase Chain Reaction Detection of Viral Genome in Childhood Pneumonia and Myocarditis

BACKGROUND Infectious respiratory disorders are important causes of childhood morbidity and mortality. Viral causes are common and may lead to rapid deterioration, requiring mechanical ventilation; myocardial dysfunction may accompany respiratory decompensation. The etiologic viral diagnosis may be difficult with classic methods. The purpose of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for identification of causative agents. METHODS AND RESULTS PCR was used to amplify sequences of viruses known to cause childhood viral pneumonia and myocarditis. Oligonucleotide primers were designed to amplify specific sequences of DNA virus (adenovirus, cytomegalovirus, herpes simplex virus, and Epstein-Barr virus) and RNA virus (enterovirus, respiratory syncytial virus, influenza A, and influenza B) genomes. Tracheal aspirate samples were obtained from 32 intubated patients and nucleic acid extracted before PCR. PCR results were compared with results of culture, serology, and antigen detection methods when available. In cases of myocarditis (n=7), endomyocardial biopsy samples were analyzed by PCR and compared with tracheal aspirate studies. PCR amplification of viral genome occurred in 18 of 32 samples (56%), with 3 samples PCR positive for 2 viral genomes. Amplified viral sequences included RSV (n=3), enterovirus (n=5), cytomegalovirus (n=4), adenovirus (n=3), herpes simplex virus (n=2), Epstein-Barr virus (n=1), influenza A (n=2), and influenza B (n=1). All 7 cases of myocarditis amplified the same viral genome from heart as found by tracheal aspirate. CONCLUSIONS PCR is a rapid and sensitive diagnostic tool in cases of viral pneumonia with or without myocarditis, and tracheal aspirate appears to be excellent for analysis.

Analysis of formalin-fixed and frozen myocardial autopsy samples for viral genome in childhood myocarditis and dilated cardiomyopathy with endocardial fibroelastosis using polymerase chain reaction (PCR)

Viral infection of the myocardium is implicated in the pathogenesis of myocarditis and dilated cardiomyopathy (DCM). Enteroviruses have been considered the most common viral etiologic agents, based on peripheral culture and serologic methods. Recently, polymerase chain reaction (PCR) has been shown to be useful in the detection of viral genomes from various infected organs and body fluids. In this study, myocardial samples from autopsy specimens (formalin fixed and fresh frozen) were examined for enteroviral and DNA viral (adenovirus, herpes simplex virus [HSV], and cytomegalovirus (CMV]) genome by PCR. The specimens studied were from 58 patients with myocarditis, 28 patients with DCM and endocardial fibroelastosis [EFE], and 22 controls. Viral genome was detectable in 34 of the 58 (59%) autopsy-proven myocarditis samples (18 adenovirus, 12 enterovirus, 2 CMV, 2 HSV) and 6 of the 28 samples from patients with DCM and EFE (6 adenovirus). We conclude that PCR is effective in the rapid amplification of virus from frozen and formalin-fixed myocardial samples and that adenovirus is an important etiologic agent in viral myocarditis as well as DCM with EFE.

ASSOCIATION OF ADENOVIRUS INFECTION WITH APOPTOSIS IN THE PATHOGENESIS OF MYOCARDITIS AND DILATED CARDIOMYOPATHY • 97

ASSOCIATION OF ADENOVIRUS INFECTION WITH APOPTOSIS IN THE PATHOGENESIS OF MYOCARDITIS AND DILATED CARDIOMYOPATHY • 97