title: Low frequency of detection by nested polymerase chain reaction of enterovirus ribonucleic acid in endomyocardial tissue of patients with idiopathic dilated cardiomyopathy.
abstract: OBJECTIVE
  The purpose of this study was to determine the prevalence of enteroviral infection in the myocardium of patients with idiopathic dilated cardiomyopathy by using a highly sensitive and specific detection technique.
  
  
  BACKGROUND
  Recent molecular studies have suggested that enteroviral persistence (in particular, coxsackieviruses type B) may underlie idiopathic myocarditis and dilated cardiomyopathy.
  
  
  METHODS
  The method used to detect enterovirus-specific ribonucleic acids (RNAs) is based on reverse transcription and nested polymerase chain reaction amplification with four pairs of primers from the conserved 5' noncoding region of the enteroviral genome. Several members of the Enterovirus genus are detectable by this assay (coxsackieviruses B1 to B6; polioviruses 1 to 3; echoviruses 9, 19 and 31), with a sensitivity threshold close to the detection of a single molecule of viral RNA in 1 mg of tissue sample. Endomyocardial tissue samples from 84 subjects were analyzed (77 samples obtained from left endomyocardial biopsies, 7 from explanted hearts). The subjects comprised 63 study patients (53 with dilated cardiomyopathy, 3 with idiopathic myocarditis, 1 with right ventricular dysplasia, 1 with restrictive cardiomyopathy, 1 with eosinophilic myocarditis, 1 with primary ventricular fibrillation and 3 with myocarditis of known etiology) and 21 control subjects with other diseases.
  
  
  RESULTS
  Positive signals were obtained only in samples from six study patients (four with dilated cardiomyopathy, one with right ventricular dysplasia and one with myocarditis). Samples from control subjects, uninfected rat myocardium and cultured cell lines yielded systematically negative results. Moreover, the nucleotide sequence analysis of the amplification products from patients with positive samples raised doubts about the true positivity of these samples.
  
  
  CONCLUSIONS
  This study suggests that the persistence of enteroviral RNA in dilated cardiomyopathy is not a major cause of the disease and that a careful analysis of polymerase chain reaction amplification products is essential in any study in which this technique is pushed to high sensitivity thresholds.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (UNIDO), Trieste, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Giacca
authors.forename: G M
authors.initials: GM
authors.lastname: Severini
authors.forename: L
authors.initials: L
authors.lastname: Mestroni
authors.forename: A
authors.initials: A
authors.lastname: Salvi
authors.forename: G
authors.initials: G
authors.lastname: Lardieri
authors.forename: A
authors.initials: A
authors.lastname: Falaschi
authors.forename: F
authors.initials: F
authors.lastname: Camerini
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D002311:Cardiomyopathy, Dilated / Q000150:complications / Q000382:microbiology*; D004770:Enterovirus / Q000235:genetics*; D004769:Enterovirus Infections / Q000150:complications / Q000453:epidemiology / Q000382:microbiology; D005260:Female; D005784:Gene Amplification; D016679:Genome, Viral; D006321:Heart / Q000382:microbiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D015995:Prevalence; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral