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Journal of Clinical Microbiology, May 2004, p. 2298-2300, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2298-2300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Frequent Detection of Viral Nucleic Acids in Heart Valve Tissue

Oliver Donoso Mantke,1* Rudolf Meyer,2 Susanna Prösch,3 and Matthias Niedrig1

Centre for Biological Safety, Robert Koch-Institut,1 German Heart Institute Berlin, 13353 Berlin,2 Institute of Virology, Charité, 10117 Berlin, Germany3

Received 6 November 2003/ Returned for modification 26 December 2003/ Accepted 10 February 2004


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ABSTRACT
 
Due to a paucity of published data concerning the prevalence of viral nucleic acid in homografts, we analyzed tissue from 30 donor hearts for the presence of viral genome sequences of enteroviruses, adenoviruses, human cytomegalovirus, and influenza virus using different PCR techniques. Viral DNA was amplified in 64 and 52% of the subvalvular myocardial tissue and non-coronary valve samples, respectively. These findings, compared with clinical history and histologic and serologic analysis, demonstrate the importance of viral safety measures in heart valve banking.


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TEXT
 
The replacement of heart valves by cryopreserved homografts is an established surgical option for patients with heart valve diseases (10). Heart valve banks accept donor hearts for homografts from different types of donors up to 65 years of age: cadaveric or non-heart-beating donors (this donor type is restricted in some member states of the European Union) and heart-beating donors, who include multiorgan donors (MOD) as well as heart transplant recipient (HTx) donors. Besides control for bacterial and fungal infections, achieved by microbial testing and antibiotic treatment of the dissected heart valves, the screening of heart tissue donors for virus infections represents another major aspect of the safety and quality assurance for homografts. Routine viral safety measures consist of careful selection of donors according to clinical status and medical history and examination of donor serum for infection with human immunodeficiency, hepatitis B, and hepatitis C viruses. However, these methods do not reliably identify acute and even persistent viral infections in the tissue donors. Molecular genetic techniques like PCR are feasible and preferable.

Cardiotropic viruses such as enteroviruses (EV), adenoviruses (AdV), human cytomegalovirus (CMV), parvovirus B19 (PVB19), and influenza virus are a great concern in heart valve banking, since all are implicated in the pathogenesis of inflammatory heart diseases like myocarditis, dilated cardiomyopathy, or coronary heart disease (1, 4). Furthermore, they can cause posttransplantation complications including graft loss (9). In a previous PCR screening of myocardial tissue samples from different sections of donor hearts (excluding subvalvular myocardium tissue and heart valve tissue), we found viral genome sequences of cardiotropic viruses (EV, CMV, and AdV) in 16 out of 50 (32%) hearts analyzed (2).

These data prompted us to investigate subvalvular myocardial tissue samples from the aortic valve (ASVM) or pulmonary valve (PSVM) and samples from the non-coronary valve (NCV) of 30 donor hearts for the presence of viral genome sequences using nucleic acid tests as described previously: PCR techniques for the detection of CMV (6) and AdV (7) and a fluorogenic reverse transcription-PCR for the detection of influenza viruses A and B (8). For the detection of EV we used a nested reverse transcription-PCR (5), and for the detection of PVB19 we used a new real-time PCR assay (3). In 16 of 30 donor hearts analyzed EV cDNA was amplified (Table 1). AdV, CMV, and parvovirus DNAs were detected in 2, 15, and 12 of 30 donor hearts, respectively. All samples were negative for influenza virus RNA. In total, 37 (64%) of the subvalvular myocardial tissue samples from 29 donors (19 from the aortic valve and 18 from the pulmonary valve) contained viral genome sequences. Remarkably, 15 of 29 (52%) NCVs analyzed so far were positive for at least one of the tested viruses. In 11 donor hearts simultaneous infection by two different viruses was observed, and in three donor hearts triple infections were observed. However, the virus load in the samples was often low, indicating a persistent viral infection.


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  		TABLE 1. Screening of heart valve donors: results from histologic, serologic, and PCR analysesa

The PCR results were compared with the clinical history, histologic analysis, and serologic status of the donors and their respective hearts (Table 1). All samples came from heart-beating donors, who were randomly selected. All of the eight HTx donors showed histologically proven, clearly defined heart disease. Of the 12 MOD hearts 5 demonstrated a normal histology while 7 were defined as hypertrophic. The 10 MOD hearts from donors >=65 years of age were also found to be hypertrophic, except one which was declared histologically normal. Thus, the majority of the donor hearts analyzed were hypertrophic and showed typical signs of left ventricular structural remodeling in the myocardium such as left ventricular dilation and wall thinning with medium to severe damage of the myocardium. These abnormalities can often be observed in chronic virus-induced heart disease (1). The data from histologic analysis and PCR matched very well: the incidence of viral genomes was higher in hypertrophic hearts than in histologically normal hearts. However, even in histologically normal donor hearts the rate of viral genome detection was certainly high, as in our previous study (2). These results indicate that histologically normal donor hearts may be more often infected or contaminated than supposed. Furthermore, it seems that there is a trend toward a higher prevalence of viral nucleic acid in subvalvular myocardium tissue and heart valve tissue than in myocardium tissue of other sections of the heart. Nevertheless, this conclusion must be interpreted carefully due to the low number of samples analyzed. The hearts of donors 3 and 26, who showed dilated cardiomyopathy or hypertrophy, were negative for viral genome sequences in ASVM, PSVM and NCV but were positive for EV cDNA in other locations of the myocardium including the septum (data not shown). EV-, AdV-, CMV-, and PVB19-specific antibodies (immunoglobulin M [IgM] and IgG) were determined for only a limited number of donors with commercial enzyme-linked immunosorbent assay kits (Table 1): EV IgM-IgG kit (Genzyme Virotech, Rüsselsheim, Germany), AdV IgM-IgG kit (BAG, Lich, Germany), CMV IgM kit (Medac, Wedel, Germany), CMV ETI-Cytok-G Plus (Sorin Biomedica), and PVB19 recomWell IgM-IgG (Mikrogen, Martinsried, Germany). The correlation between serologic and PCR data for some viruses was relatively low, indicating that serology alone is not a reliable method for detecting acute or persistent cardiotropic viral infections of the heart. For example, of nine donors positive for EV cDNA only three were positive for EV IgM or IgG.

In summary, the results obtained show that viral genome sequences are frequently present in heart valve tissues. Although the PCR data are not able to define whether there is infectious virus in the heart tissue, they indicate that there is a risk for transmission of viral infection by heart valve transplantation. Besides the routine selection of donors and examination of donor serum for viral infection, we suggest that screening of heart valve tissue by PCR should be introduced into heart valve banking, at least for known cardiotropic viruses such as EV, AdV, CMV, and PVB19. To evaluate the risk of infection in recipients, long-term follow-up studies are necessary. Careful screening for acute or chronic infections in donors and recipients by PCR will facilitate decisions about selection of donor organs and tissues. Furthermore, the use of PCR as a screening tool in antiviral therapies constitutes an option to improve treatment regimens that could limit inflammatory damage of the heart after transplantation.


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ACKNOWLEDGMENTS
 
This work was supported by the Robert Koch-Institut doctoral fellowship program.

We thank Anette Teichmann, Ingrid Zadow, Ernie Schmitzer, and Karin Muske for their excellent technical assistance. In addition we thank Kim Hattermann, Hi-Gung Bae, and Stephen Norley for critically reading the manuscript and for helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Biological Safety, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany. Phone: 49-30-4547-2244. Fax: 49-30-4547-2604. E-mail: donosoo{at}rki.de. Back


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Journal of Clinical Microbiology, May 2004, p. 2298-2300, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2298-2300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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