Detection Of Viral Genomes In The Heart By Rtpcr

In the last decade, another highly sensitive technique, the PCR, has become the most commonly used method for the detection of viral DNA and RNA sequences in the heart of patients with myocarditis and DCM. Primarily as a result of the identification of different viral nucleic acids in the myocardium of these patients by PCR, it is widely thought that myocarditis can develop as a result of infection with enteroviruses,4 adenoviruses,5'6 cytomegalovirus,6'9 Epstein-Barr virus,12 herpes simplex virus,6 human herpesvirus-6,13 HIV,14 influenza A and influenza B virus,18 PVB19,15-17 or hepatitis C virus.21,22 Martin et al.5 reported in a PCR analysis of children with acute myocarditis that viral sequences were present in 26 of 38 myocardial samples (68%): 8 enterovirus, 15 adenovirus, 2 herpes simplex virus, and 1 cytomegalovirus. In addition, PCR analysis of endomyocardial biopsy specimens suggests that fibroelastosis is a sequela of viral myocarditis after infection with mumps virus (> 70% positive samples) and adenovirus (28% positive samples).19

Regarding the presence of infectious agents in the heart of patients with DCM, there are rather divergent PCR results. Whereas in patients with DCM Pankuweit et al.9 found an incidence of cytomegalovirus DNA of 12%, adenoviral DNA of 15%, and borreliosis of 0.5%, Galama's group did not detect any nucleic acids from enteroviruses, cytomegalovirus, hepatitis B and C viruses, Borrelia burgdorferi, Chlamydia species, mycoplasmata, or Toxoplasma gondii in patients with end-stage DCM.94

PCR provides a fast and sensitive method to detect microbial nucleic acid sequences in cardiac tissue. However, a major disadvantage of the PCR technique is the failure to differentiate among infected cell types, and detection of virus in endomyocardial biopsy specimens might reflect accidental amplification of viral sequences in persistently infected blood cells. Thus, PCR results provide only circumstantial evidence for a pathogenic role of several infectious agents in suspected myocarditis. Further substantiation by in situ hybridization and exclusion of systemic infections is needed.

However, there is firm evidence for a causal link between myocarditis and DCM with acute and persistent enterovirus infection not only from findings in the murine models of enteroviral heart disease95-98 but also from in situ hybridization studies in children and adults.26'28'57'64'65'67'99 Unlike other infectious agents, the importance of enteroviruses in the development of heart diseases was proved by visualizing the cytotoxicity of enterovirus replication in myocytes and the consequent invasion of the reactive immune response, leading to acute and chronic myocarditis. Because the in situ hybridization technique is not available in all laboratories, the widely used PCR method was also introduced to diagnose enteroviral myocarditis.

For the identification of enteroviral sequences in clinical material, various PCR protocols have been developed. Generally, the detection of enteroviral RNA in fresh-frozen or formalin-fixed and paraffin-embedded100-105 heart muscle tissue by RT-PCR requires several steps: 1) isolation of viral RNA from tissue, 2) reverse transcription of RNA into cDNA, 3) amplification of a specific region of the cDNA by PCR and application of enterovirus-specific primers and a heat-stable DNA polymerase, 4) confirmation of specificity by nested PCR or Southern blot hybridization, and 5) sequencing of PCR products. Most reports describe the use of enterovirus-specific primers from the well-conserved 5' non-coding region of the enteroviral RNA, which are capable of detecting a majority of enteroviral serotypes (Fig. 13-8). Because of the high number of potentially cardiotropic serotypes, serotype-specific PCRs are usually not practical for clinical purposes. If, however, the identification of genotypes of enteroviruses is required, a PCR can be performed using primers covering the enterovirus VP2 region106'107 in addition to single-strand conformation bp

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