Parvovirus B19 in Endomyocardial Biopsy of Patients With Idiopathic Dilated Cardiomyopathy: Foe or Bystander?

BACKGROUND: Parvovirus B19 (PVB19) has emerged as one of the viruses possibly inducing chronic myocarditis and subsequent idiopathic dilated cardiomyopathy (IDCM). The aim of this work was to investigate the presence and long-term consequences of PVB19-DNA within myocardial biopsies from patients with IDCM and to compare the findings with those from donor hearts (control group). METHODS AND RESULTS: Forty hospitalized IDCM patients (age 47 ± 12 y) with mean left ventricular ejection fraction 27 ± 12% were included. The presence of PVB19-DNA in myocardial biopsies and of IgG and IgM antibodies in patient sera was analyzed. The control group consisted of 20 donor hearts. The follow-up time was 112 ± 57 months. PVB19-DNA was found in myocardial biopsies of both patients (73%) and control samples (55%; P = .25).Three deaths and 8 heart transplantations occurred in the IDCM group, and 6 deaths in the control group (ie, the recipients of the control hearts). No difference in transplantation-free survival between the PVB19-DNA positive/negative IDCM patients or transplant recipients was found. CONCLUSIONS: PVB19-DNA is a common finding in both patients with IDCM and in healthy donor hearts, not affecting prognosis. These findings support the view that PVB19 is an innocent bystander, frequently found in myocardium with low DNA copies, and not a plausible cause of IDCM.

Viral endomyocardial infection in the 1st year post transplant is associated with persistent inflammation in children who have undergone cardiac transplant.

BACKGROUND: Viral genome in cardiac allograft has been associated with early graft loss in children who have undergone cardiac transplant from unknown mechanisms. METHODS: This study is a retrospective review of children who have undergone cardiac transplant at a single institution from 1/2004 to 5/2008. Patients underwent cardiac catheterisations with endomyocardial biopsies to evaluate for rejection--graded on Texas Heart Institute scale--and the presence of virus by polymerase chain reaction. Patients with virus identified during the first year post transplant were compared at 1 year post transplant with virus-free patients. RESULTS: The cohort comprised 59 patients, and the median age at transplant was 5.1 years. Viral genomes were isolated from 18 (31%) patients. The PCR + group had increased inflammation on endomyocardial biopsies, with a median score of 4 (ISHLT IR) versus 1 (ISHLT 1R) in the PCR--group (p = 0.014). The PCR + group had a similar cardiac index (median 3.7 ml/min/m(2)), pulmonary capillary wedge pressure (median 10 mmHg), and pulmonary vascular resistance index (median 1.7 U m(2)) comparatively. PCR + patients were more likely to have experienced an episode of rejection (p = 0.004). CONCLUSIONS: Children who developed viral endomyocardial infections after a cardiac transplant have increased allograft inflammation compared with virus-free patients. However, the haemodynamic profile is similar between the groups. The ongoing subclinical inflammation may contribute to the early graft loss associated with these patients.

Idiopathic dilated cardiomyopathy and persistent viral infection: lack of association in a controlled study using a quantitative assay.

BACKGROUND: It remains unclear whether idiopathic dilated cardiomyopathy (DCM) might ensue as the consequence of viral myocarditis, due to viral persistence in cardiomyocytes. To address this issue, we quantified the levels of enterovirus, Epstein-Barr virus (EBV), Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2), adenovirus and parvovirus B19 genomes in endomyocardial biopsies (EMBs) from patients with DCM, active myocarditis and controls. METHODS: Real-time polymerase chain reaction (PCR)-based methods using TaqMan probes were developed for the quantitative detection of viral genomes in EMBs from 35 patients with DCM and 17 with active myocarditis. A control group included 20 surgical patients with valve or coronary artery disease. RESULTS: None of the 72 samples tested positive for enteroviruses, EBV, HSV-1 or -2. One DCM patient tested positive for adenovirus. Of notice, 20/52 (38%) of patients with cardiomyopathy and 8/20 (40%) of controls were positive for parvovirus B19; no significant differences in viral titre were detected between groups. CONCLUSIONS: Our preliminary results disfavour the hypothesis that persistent myocardial viral infection might be a frequent cause of DCM. The detection of parvovirus B19 from both cardiomyopathy and non-cardiomyopathy patients supports the notion that this virus is widely spread in the population.

Evolution of the inflammatory response in vertebrates: fish TNF-alpha is a powerful activator of endothelial cells but hardly activates phagocytes.

TNF-alpha is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-alpha are mediated through the activation of endothelial cells. Thus, TNF-alpha promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-alpha, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-alpha in Ag presentation. Lastly, we found that TNF-alpha increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-alpha on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-alpha increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-alpha resulted in increased viral replication. Together, these results indicate that fish TNF-alpha displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Immunohistological detection of Parvovirus B19 capsid proteins in endomyocardial biopsies from dilated cardiomyopathy patients.

BACKGROUND: Parvovirus B19 (B19V) has been identified as the most common virus in dilated cardiomyopathy (DCM) patients by PCR techniques. We investigated the detectability and the expression pattern of B19V proteins by immunohistology (IH) in endomyocardial biopsies (EMBs) from DCM patients, and its association with the standard proof of B19V genomes by nested PCR (nPCR. MATERIAL/METHODS: EMBs from 30 DCM patients were analyzed by nPCR for B19V genomes, and IH of B19V VP1/VP2 expression was carried out using the antibody clone R92F6. The specificity of this antibody was investigated on 293T cells transfected with pB19-M20. Positive anti-B19V IH staining (positive in n=14/46.6%) and nPCR proof of B19V genomes (positive in n=15/50%) were significantly associated (p=0.0003). RESULTS: Based on the B19V nPCR results, the sensitivity of anti-B19V-VP1/VP2 IH was 80.0%, and the specificity was 86.0%. B19V immunostaining was observed on interstitial cells in all IH positive cases, and was noted additionally on endothelial cells in 1, and on cardiomyocytes in 4 cases. CONCLUSIONS: IH detection of B19V VP1/VP2 expression provides a high association with the nPCR proof of B19V genomes in DCM patients. IH detection of B19V proteins enables a differentiation of B19V VP1/VP2 expressing cells within the myocardium.

[Reversible dilated cardiomyopathy in a patient with acute, advanced heart failure and intense endothelial inflammatory reaction in endomyocardial biopsy--a case report]. / Odwracalna kardiomiopatia rozstrzeniowa u chorego z ostra i ciezka niewydolnoscia serca oraz nasilona odpowiedzia zapalna sródblonka w biopsji endomiokardialnej.

Inflammatory response of the endothelium has been increasingly recognized in the aetiopathogenesis of sporadic dilated cardiomyopathy (DCM). It has been shown that up to 2/3 of patients with DCM have immunohistological evidence of enhanced activation of the endothelium. We present a case of a middle-aged patient with a history of hypertension and hyperlipidaemia who developed sudden significant left ventricular dysfunction following flu-like syndrome. Endomyocardial biopsy revealed no myocarditis, but immunohistological features of endothelial activation were present. Additionally, increasing titers of IgG antibodies against PvB19 were observed. During 18 months of standard heart failure treatment along with statin therapy, we observed a significant recovery of left ventricular systolic function, and in this way, reversible dilated cardiomyopathy.

Overview on chronic viral cardiomyopathy/chronic myocarditis.

Myocarditis is most often induced by cardiotropic viruses and often resolves with minimal cardiac remodelling and without discernable prognostic impact. Acute myocarditis has a highly diverse clinical presentation (asymptomatic, infarct-like presentation, atrioventricular (AV)-block, atrial fibrillation, sudden death due to ventricular tachycardia, fulminant myocarditis with severely depressed contractility). Progression of myocarditis to its sequela, dilated cardiomyopathy (DCM), has been documented in 20% of cases and is pathogenically linked to chronic inflammation and viral persistence. Persistence of cardiotropic viruses (enterovirus, adenovirus) constitutes one of the predominant aetiological factors in DCM. Additionally, circulating autoantibodies to distinct cardiac autoantigens have been described in patients with DCM, providing evidence for autoimmune involvement. Since clinical complaints of myocarditis and DCM are unspecific, a positive effect of any specific therapy depends on an accurate biopsy-based diagnosis and characterization of the patients with histological, immunohistological and molecular biological methods (PCR), which have developed into sensitive tools for the detection of different viruses, active viral replication, and myocardial inflammation. The immunohistochemical characterization of infiltrates has supported a new era in the diagnosis of myocardial inflammation compared with the Dallas criteria, which has led to a new entity of secondary cardiomyopathies acknowledged by the WHO, the inflammatory cardiomyopathies (DCMi). Immunohistochemically quantified lymphocytes significantly better reflect troponin levels and correlate with findings by anti-myosin scintigraphy compared with the histological analysis. Furthermore, the orchestrated induction of endothelial cell adhesion molecules (CAMs) in 65% of DCM patients has confirmed that CAM induction is a prerequisite for lymphocytic infiltration in DCMi. The combination of these immunohistological with molecular biological diagnostic techniques of virus analysis allows a further classification of dilated cardiomyopathy by differentiating the disease entity in subgroups of virus-positive and virus-negative patients with or without cardiac inflammation. Further analysis of the predominant Th1-/Th2-immune response may provide additional prognostic information on the natural course of the disease. This differential analysis improves the clinical management of patients and is an indispensable prerequisite for the development of specific antiviral or immunomodulatory treatment strategies.

High prevalence of viral genomes and inflammation in peripartum cardiomyopathy.

Molecular pathologic investigation of endomyocardial biopsy specimens from 26 patients with peripartum cardiomyopathy revealed viral genomes (parvovirus B19, human herpes virus 6, Epstein-Barr virus, and human cytomegalovirus) in 8 patients (30.7%) that were associated immunohistologically with interstitial inflammation. These findings indicate a high prevalence of virus-associated inflammatory changes in peripartum cardiomyopathy.

Escape from cardiomyocyte apoptosis by enterovirus persistence due to elevated soluble Fas-receptors.

BACKGROUND: Apoptosis causes loss of contractile cardiomyocytes in inflammatory heart disease. Despite recent examinations, the influence of virus infection on apoptosis remained ill-defined. METHODS: Apoptosis was assessed in left ventricular endomyocardial biopsies by the TUNEL method frompatients with chronic myocarditis and adeno-, cytomegalo- and enterovirus persistence. Soluble Fas-ligands, sFas-receptors, TNF-alpha, IL-6, IL-10 and IFN-gamma were measured using ELISA technique. RESULTS: Elevated (P < 0.05) rates of apoptosis were found in patients with autoimmune myocarditis. Apoptosis was increased (P < 0.05) in the case of cytomegalovirus persistence, but not significantly increased in the presence of adenoviral genome. No evidence for apoptosis, but elevated concentrations of soluble Fas-receptors were found only in the case of enterovirus persistence. In turn, elevated percentages of apoptosis and normal soluble Fas-receptor concentrations were found in patients with chronic myocarditis. Serum levels of soluble Fas-ligands, TNF-alpha, IL-6, IL-10 and IFN-gamma did not predict changes in TUNEL-positivity. CONCLUSIONS: Escape mechanisms to protect cardiomyocytes from apoptosis are yet not known for enterovirus infections. Soluble Fas-receptors have to be considered to counteract binding of soluble Fas-ligands that results in the blockade of apoptosis induction. It is a new finding that soluble Fasreceptors were elevated in the presence of enterovirus genome in the heart. Inhibition of apoptosis can impair virus clearing and prolong its replication with a potential worse outcome. In turn, sufficient protection from apoptosis in autoimmune myocarditis should reduce loss of cardiomyocytes. Therefore, the interaction of the Fas components could provide a new therapeutic target in myocarditis.

Frequency and quantity of the parvovirus B19 genome in endomyocardial biopsies from patients with suspected myocarditis or idiopathic left ventricular dysfunction.

Parvovirus B19 (PB19) has been identified as a possible cause of myocarditis and heart failure in both children and adult patients. This study used real time PCR analysis, to determine the frequency and to quantify PB19 viral genomes in endomyocardial tissue samples from 80 adult patients with clinically suspected myocarditis or idiopathic left ventricular dysfunction and from 36 controls. Histological (Dallas classification) and immunohistological analyses were performed to detect myocardial inflammation in the endomyocardial biopsies.PB19 genomic DNA was found in nine of 80 patients (11.2%), 4 out of 31 (12.9%) patients with inflammatory infiltrates detected via immunohistological methods and 5 out of 49 (10.2%) patients with left ventricular dysfunction without myocardial inflammation. The copy numbers for PB19 DNA ranged between 30 and 3900 per microg of cellular DNA. Four patients with clinically suspected myocarditis had copy numbers for PB19 DNA of 70, 740, 3400 and 3900, respectively, per microg of cellular DNA in the endomyocardial biopsy. Five patients with idiopathic left ventricular dysfunction had copy numbers for PB19 DNA of 30, 38, 52, 58 and 90, respectively, per microg of cellular DNA in the endomyocardial biopsy. The amplicon of one of the nine positive PCR fragment was sequenced and was found to be fully identical in the highly conserved sequence of published Parvovirus B19 VP1/VP2 genes (NCBI gene bank). In all patients, acute myocarditis was excluded according to the Dallas classification. All biopsies of 36 controls with no history of myocarditis or recent viral infection were negative for myocardial inflammation and parvovirus B19 genomes. In summary, Parvovirus B19 DNA is present within the myocardium of patients with suspected myocarditis and idiopathic left ventricular dysfunction and can be detected and quantified in endomyocardial specimens via real time PCR.

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.

Frequent detection of parvovirus B19 genome in the myocardium of adult patients with idiopathic dilated cardiomyopathy.

Aside from enteroviruses and other viruses, e.g., adenoviruses, which are known to be associated with idiopathic dilated cardiomyopathy (IDC), a cardiac tropism is also attributed to parvovirus B19 (PVB19). The purpose of the present study was to determine the prevalence of enterovirus, adenovirus and PVB19 genomes in the myocardium of adult patients with IDC and to analyze the significance of PVB19 with regard to the course of the disease, as compared to the other cardiotropic viruses. In 52 adult patients with IDC and 10 control patients with normal left ventricular ejection fraction (> or =55%) undergoing coronary artery bypass surgery, myocardial tissue samples were investigated for enteroviral RNA using polymerase chain reaction (PCR) and Southern blot hybridization of the PCR product. Specific nested PCR was used to assess the prevalence of adenovirus and PVB19 DNA, in addition to sequencing of the latter. The clinical and echocardiographic course of the disease was followed for a mean (+/- SD) period of 21.1+/-9.5 months. Fourteen of the 52 patients (27%) were enterovirus-positive, 2/52 (4%) patients were adenovirus-positive, 14/52 (27%) patients were PVB19-positive, 8/52 (15%) patients were enterovirus plus PVB19-positive, and in 14/52 (27%) patients no viral genomes were found. Six patients died during the follow-up period, without any significant difference between the patient groups: 1/14 (7%) in the enterovirus-positive, 0/2 (0%) in the adenovirus-positive, 2/14 (14%) in the PVB19-positive, 1/8 (12.5%) in the enterovirus plus PVB19-positive, and 2/14 (14%) in the virus-negative group. PVB19 genome was found in 4 of the 10 (40%) control patients, but no enterovirus or adenovirus genomes were detected in these patients. In conclusion, in the myocardium of patients with IDC, PVB19 is detectable as frequently as enteroviral genome. PVB19-positive patients with IDC have a rather favorable prognosis and do not differ significantly from the other virus-positive or virus-negative patient groups with respect to survival. Finally, the pathogenetic and prognostic significance of PVB19 in IDC still remains unclear.

Prevalence of the parvovirus B19 genome in endomyocardial biopsy specimens.

Although enteroviruses have long been considered the most common cause of inflammatory heart muscle diseases, parvovirus B19 (PVB19) is emerging as a new and important candidate for myocarditis and dilated cardiomyopathy with inflammation (DCMi) and without inflammation (DCM). We investigated left ventricular endomyocardial biopsy specimens from 110 patients with suspected inflammatory heart disease for the presence of PVB19, Coxsackie virus (CVB), and adenovirus (Ad2) genome by polymerase chain reaction. Diagnosis of myocarditis (36 patients), DCM (18 patients), DCMi (13 patients), and perimyocarditis (12 patients) was made by immunohistochemical and histopathological investigation of endomyocardial biopsy specimens. A control group consisting of patients with arterial hypertension was also investigated. Prevalence of the PVB19 genome in endomyocardial biopsy specimens was highest in patients with DCMi (3 of 13) and patients with myocarditis (7 of 36); in patients with DCM and perimyocarditis, prevalence was 3 of 13 and 2 of 12, respectively. In patients with resolved myocarditis, no PVB19 DNA was detected; in patients with no inflammation and controls, prevalence was only 4% and 7%, respectively. CVB-RNA was detected in endomyocardial biopsy specimens from 3 of 37 patients with myocarditis; Ad2-DNA was found in 1 patient with DCM and 1 patient with perimyocarditis. These findings suggest an association of the PVB19 genome in endomyocardial biopsy specimens of adults with the development of DCM, DCMi, and chronic myocarditis more frequently than previously expected. PVB19 should therefore be recognized as a potential cardiotropic pathogen in patients of all ages.

Molecular diagnosis of myocarditis and dilated cardiomyopathy in children: clinicopathologic features and prognostic implications.

Myocarditis is the most common cause of heart failure in children. We investigated viral etiology of myocarditis/dilated cardiomyopathy (DCM) in children and correlated molecular findings with pathologic and clinical data. Polymerase chain reaction (PCR) or reverse transcription (RT)-PCR were used to analyze 59 endomyocardial biopsies from 48 consecutive young (<18 yrs) patients (pts) with clinical and histologic diagnosis of myocarditis and DCM, employing primers designed to amplify specific sequences of various DNA and RNA viruses. Nucleic acids were successfully extracted in 41 pts and viral genomes were found in 20 (49%): 12 out of 26 pts (46%) with myocarditis, 6 out of 13 (46%) pts with DCM, and both patients with endocardial fibroelastosis. Enteroviruses were more common in DCM (72%), whereas adenoviruses and enteroviruses shared the same rate (36%) in myocarditis. The mumps virus genome was detected in the two pts with endocardial fibroelastosis. More diffuse inflammatory infiltrates and myocyte damage as well as more impaired left ventricular end diastolic volume and shortening fraction were noted in viral positive cases. PCR positive pts had a worse outcome, resulting in transplantation or death. Three out of 8 pts with viral myocarditis who underwent cardiac transplantation had recurrent PCR-proven graft viral infection. Viral myocarditis/DCM appeared to be a more severe disease than nonviral forms. Enteroviruses were more common in DCM, whereas adenoviruses were as frequent as enteroviruses in myocarditis. Persistence of viral infection was associated with disease deterioration. Viral myocarditis relapsed after transplantation.

[Parvovirus B19 myocarditis in a young man with previous non-bacterial meningitis]. / Parvovirus B19-Myokarditis eines jungen Erwachsenen nach abakterieller Meningitis.

HISTORY AND CLINICAL FINDINGS: A 22-year old man presented with fatigue, dyspnea NYHA III and presyncopes that had persisted since a non-bacterial meningitis 3 months before. INVESTIGATIONS: Transthoracic echocardiography revealed a dilated left ventricle with an ejection fraction (EF) reduced to 35-40 % due to global hypokinesia. No pericardial effusion was seen; ECG and lung function test were normal. Serological, immunological and microbiological tests as well as nested PCR analysis of blood leucocytes for detection of cardiotropic pathogens were inconclusive. In endomyocardial biopsies retrieved from the left ventricular posterolateral wall, a chronic macro-phage-rich myocarditis was shown by histopathology and, in addition, Parvovirus B19 was identified as specific pathogen by use of nested PCR analysis. TREATMENT AND COURSE: At physical rest and with ACE inhibitor therapy (2.5 mg ramipril/day), heart failure decreased steadily. Follow-up echocardiography 1 month later revealed a left ventricle that was only slightly dilated with an EF of 50 %. 3 months later, the patient was markedly more load-bearing; the EF amounted to 55-60 %. CONCLUSIONS: Parvovirus B19 should be regarded as potential pathogen in case of suspected myocarditis in adulthood. Whether the previous non-bacterial meningitis was also attributable to this specific pathogen, remains open. Of note, however, the present case report by demonstrating a localized myocardial Parvovirus B19 infection without detectable systemic infection underscores the importance of molecular tests for diagnostic accuracy in manifest organ failure.

Myocarditis mimicking acute myocardial infarction: role of endomyocardial biopsy in the differential diagnosis.

OBJECTIVE: To test the hypothesis, using endomyocardial biopsies, that unexplained cases of apparent acute myocardial infarction were caused by myocarditis. MATERIAL: Between 1992 and 1998, 12 patients were admitted to the coronary care unit with severe chest pain, ST segment elevation, increased serum creatine kinase and MB isoenzyme, and with wall motion abnormalities on echocardiogram highly suggestive of acute myocardial infarction. These patients were further investigated by endomyocardial biopsy, as their coronary angiograms were normal. A diagnosis of myocarditis was made according to the Dallas criteria. A panel of antibodies was used for immunohistochemical characterisation of inflammatory cell infiltrate. Polymerase chain reaction (PCR) was used to detect viral genomes in seven cases. RESULTS: Haematoxylin and eosin staining of the endomyocardial biopsy showed active myocarditis in six patients and borderline myocarditis in one. Immunohistochemistry was positive for inflammatory cell infiltrates in 11 patients, including all the seven who were positive on haematoxylin and eosin staining according to the Dallas criteria. Only one patient had no evidence of inflammation. PCR was positive in two patients, both for Epstein-Barr virus. Follow up showed complete resolution of echocardiographic abnormalities in all patients except one. CONCLUSIONS: Myocarditis can mimic acute myocardial infarction in patients with angiographically normal coronary arteries, leading to errors of treatment. In patients with apparent myocardial infarction and a normal coronary angiogram, endomyocardial biopsy may help in the diagnosis of myocarditis. The sensitivity of endomyocardial biopsy was enhanced by using immunohistochemical and molecular biological techniques.