DETECTION OF ENTEROVIRAL RNA IN ENDOMYOCA RDIAL BIOPSIES OF PATIENTS WITH INFLAMMATORY CARDIOMYOPATHY AND IDIOPATHIC DILATED CARDIOMYOPATHY

The role of enteroviral myocardial infection in the development of dil ated cardiomyopathy could only be substantiated after the introduction of molecular biological techniques (polymerase chain reaction, in-sit u hybridization) in virological diagnostics of dilated cardiomyopathy. By using histological and especially immunohistological techniques fo r the detection of myocardial inflammation in patients with the tentat ive clinical diagnosis of dilated cardiomyopathy, a differentiation be tween inflammatory cardiomyopathy and idiopathic dilated cardiomyopath y on the basis of the WHO classification 1995 (31) was made. Inflammat ory cardiomyopathy is defined by myocarditis in association with cardi ac dysfunction and is diagnosed by established histological and especi ally immunohistological techniques. The combination of histological, i mmunohistological, and molecularbiological techniques enabled a subgro up analysis of the incidence of enteroviral myocardial RNA in patients with inflammatory cardiomyopathy in comparison to patients with idiop athic dilated cardiomyopathy. The study involved a total of 75 patient s with impaired left ventricular function (EF < 50%) and the tentative clinical diagnosis of dilated cardiomyopathy. Right ventricular endom yocardial biopsies were obtained from all patients for further clarifi cation of the cause of left ventricular functional disorder. All biops ies were analyzed for the presence of acute and chronic inflammatory m yocardial alterations by histological (,,Dallas'' criteria) and immuno histological techniques (lymphocytic infiltrates, MHC antigen expressi on). Furthermore, each biopsy was examined by reverse transcriptase po lymerase chain reaction (RT-PCR) in combination with Southern blot hyb ridization for the presence of enteroviral RNA. Active myocarditis was excluded in all patients by histological examination according to the ''Dallas'' criteria. Using immunohistological techniques, 26/75 patie nts (35%) had evidence for chronic inflammatory myocardial alterations in the sense of lymphocytic infiltrates (greater than or equal to 2.0 CD3 T-lymphocytes/ visual field at 400 magnification (HPF); greater t han or equal to 7 CD3 T-lymphocytes/mm(2)). These patients were diagno sed as having inflammatory cardiomyopathy. To differentiate between pa tients with and without myocardial inflammation, cases with focal cell ular infiltration and an average cell number between 1.5 and 2.0 CD3 T -lymphocytes/HPF and an increased expression of additional immune mark ers, i. e., MHC antigens, were not addressed in the group of patients with inflammatory cardiomyopathy. This is in contrast to Kuhl et al. ( 19). Consequently these patients were classified as patients with idio pathic dilated cardiomyopathy. These criteria of diagnosing myocardial inflammation were based on published results (20, 23, 26, 27, 49) and on our own control group (n = 85) (19) in which mean CD3 T-lymphocyte count/HPF in normal myocardial tissue were 0.7 (range 0.0-1.4). In ad dition, a subgroup analysis was performed of patients with a CD3 T-lym phocyte count greater than or equal to 3 CD3 T-lymphocytes/HPF (greate r than or equal to 11 CD3 T-lymphocytes/mm(2)). The other 49/75 patien ts without myocardial inflammation (< 2.0 CD3 T-lymphocytes/HPF) were diagnosed as having idiopathic dilated cardiomyopathy. In 27/75 patien ts (36%), RT-PCR in combination with Southern blot hybridization revea led enteroviral RNA in the endomyocardial biopsies. The detection rate of enteroviral RNA did not differ between inflammatory cardiomyopathy (8/26 (31%)) and idiopathic dilated cardiomyopathy (19/49 (39%)). In the subgroups of patients with a CD3 T-lymphocyte cell count greater t han or equal to 3 CD3 T-lymphocytes/HPF (greater than or equal to 11 C D3 T-lymphocytes/mm(2)) (mean 4.4 +/- 2.1 CD3 T-lymphocytes/HPF), thre e of the ten patients were enteroviral RNA positive (30%). In summary, the introduction of histological and immunohistological techniques in the extended diagnostics of dilated cardiomyopathy enables a subgroup analysis of the incidence of enteroviral myocardial RNA in inflammato ry and idiopathic cardiomyopathy. This advanced diagnostic approach re vealed a comparable incidence of enteroviral RNA in patients with infl ammatory cardiomyopathy (8/26) and idiopathic dilated cardiomyopathy ( 19/49. In the subgroup of patients with greater than or equal to 3 CD3 T-lymphocytes/HPF (n = 10) 3/10 patients (30%) were also enterovirus positive. Interestingly, the highest incidence of enteroviral RNA dete ction (39%) could be documented in patients with idiopathic dilated ca rdiomyopathy, i.e., without any evidence of myocardial inflammation. H owever, to what extent inflammation-induced myocardial alterations not observed in this disease have an impact on viral persistence has to b e investigated in further studies. It is the task of future clinical t rials to show whether these findings will have an impact on further ri sk stratifications and the development of future therapeutic options.