Section 3 -

Diagnostic Virology of EBV and Adenovirus in Transplant Recipients Randall T. Hayden

Immunocompromised patients are subject to a variety of infections which may produce marked differences in epidemiology, morbidity and mortality, compared to immunocompetent individuals. Disease spectrum and severity may depend heavily on a patient's underlying medical condition, the cause and degree of immunosupression. Viral infection represents a primary concern in such populations, with particularly increased frequency and consequences of infection among those with diminished cell-mediated immunity. Thus, patients with primary or secondary immune deficiencies may be at great risk for symptomatic and often severe infection with a wide array of viral pathogens. While the spectrum of viral infections in this group is broad, many laboratory diagnostic challenges are shared among them. Most such issues can be well-illustrated with only a few examples. Epstein-Barr virus (EBV) and adenovirus (ADV) will be discussed in this context. EBV 90-95% adult seropositivity rate worldwide Spread by oropharyngeal secretions Infects B lymphocytes Associated with several forms of neoplasia, including post-transplant lymphoproliferative disorder (PTLD) Associated with allogeneic transplantation and immunosuppression Mortality approaching 90% in some series Risk Factors ADV Non-enveloped DNA viruses 51 serotypes, six subgroups Variable homology within and between subgroups Increasing association with disease, mortality in immunocompromised individuals, particularly BMT patients hemorrhagic cystitis, enteritis, hepatitits, encephalitis, pneumonitis Prevalence of ADV 5-37% Invasive disease in 30-80% Risk factors Laboratory Diagnostics – Challenges Sensitivity Specificity Quantification Speed of detection Cost Practicality Clinical Correlation End-Organ Diagnosis While the identification of adenovirus in clinically infected tissues can be accomplished by culture-based methodologies, as well as by direct morphologic evidence of the infectious agent (i.e., viral inclusions), the identification of Epstein-Barr Virus in such specimens is largely based on histopathologic characterization of typical lymphoproliferative processes, in conjunction with techniques such as immunohistochemistry or in situ hybridization. In the case of adenovirus, findings include typical histopathologic evidence of infection, most often described in hepatic or pulmonary lesions. These lesions variably include features such as necrosis, epithelial hyperplasia, and inflammatory infiltrates. The latter may of course vary depending on the degree of immunosuppression in a given case. Intra-nuclear inclusions are usually present and are typically amphophilic or basophilic and smudgy in appearance. Diagnosis may be confirmed through the use of either immunohistochemistry or in situ hybridization. Adenovirus may also be isolated from various specimens using conventional cell culture methods, with the production of characteristic cytopathic effects and confirmation of diagnosis by immunoflourescent methods. These techniques are specific, but have limited sensitivity and may require prolonged incubation of culture for definitive results. Antigen detection methods are more rapid, but may also exhibit limited sensitivity. Epstein Barr Virus, as noted above, may provoke a lymphoproliferative process, hyperplasic or neoplastic in nature, without direct morphologic evidence of viral proliferation. The virus may be specifically detected in characteristic lesions using either immunohistochemistry or in situ hybridization. Immunohistochemistry targets include Epstein Barr Virus nuclear antigen (EBNA), latent membrane protein 1 (LMP-1), or Epstein-Barr-encoded RNAs (EBERs). In situ hybridization can also target one of several sequences, including EBNA, the BamH1 W repetitive sequence, or EBERs. Viral isolation from EBV-related lesions is not often performed, requiring co-cultivation of the tissue of interest together with umbilical cord lymphocytes. This procedure is of limited practicality in the clinical laboratory and its sensitivity may also be suboptimal. Detection of Systemic Infections While the detection of EBV and Adenovirus can be accomplished in the setting of clinically or histopathologically evident end-organ infection, this may represent a relatively late stage of disease. Systemic detection of these viruses, through routine surveillance techniques, may allow preemptive action at an earlier stage, with a greater potential for preventing serious or life-threatening illness. The tools available for such testing include all of the usual laboratory methodologies. However, traditional, phenotypic techniques, such as viral culture, are often too insensitive to detect the concentrations of virus circulating in the early phases of active infection. Nucleic acid amplification methods, such as PCR, hold great promise for the early detection of infection, as well as for the assessment of treatment efficacy and the determination of therapeutic endpoints. The remainder of the discussion will deal with the use of molecular methodologies including their strengths and limitations, and the particular advantages of real-time target amplification methods. Also discussed will be assay selection, design, and verification of performance, as well as ongoing assay performance and the use of test results in projecting clinical disease. Reference List Conner, D. H., F. W. Chandler, D. A. Schwartz, H. J. Manz, and E. E. Lack. 1997. Pathology of Infectious Diseases, p. 1707. Appleton&Lange, Stamford, Connecticut. Gu, Z., S. W. Belzer, C. S. Gibson, M. J. Bankowski, and R. T. Hayden. 2003. Multiplexed, real-time PCR for quantitative detection of human adenovirus. J.Clin.Microbiol. 41:4636-4641. Ison, M. G. 2006. Adenovirus infections in transplant recipients. Clin.Infect.Dis. 43:331-339. Kojaoghlanian, T., P. Flomenberg, and M. S. Horwitz. 2003. The impact of adenovirus infection on the immunocompromised host. Rev.Med.Virol. 13:155-171. Leen, A. 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